G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
protein kinase C, beta
G00000877 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000073497 (Vega human gene)
ENSG00000166501 (Ensembl human gene)
5579 (Entrez Gene)
56 (G2Cdb plasticity & disease)
PRKCB1 (GeneCards)
176970 (OMIM)
Marker Symbol
HGNC:9395 (HGNC)
Protein Expression
3843 (human protein atlas)
Protein Sequence
P05771 (UniProt)

Diseases (1)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000137: Diabetic nephropathy N Y (12874455) Single nucleotide polymorphism (SNP) Y


  • Identification of a common risk haplotype for diabetic nephropathy at the protein kinase C-beta1 (PRKCB1) gene locus.

    Araki S, Ng DP, Krolewski B, Wyrwicz L, Rogus JJ, Canani L, Makita Y, Haneda M, Warram JH and Krolewski AS

    Section on Genetics and Epidemiology, Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA.

    Abnormal activation of protein kinase C-beta isoforms in the diabetic state has been implicated in the development of diabetic nephropathy. It is thus plausible that DNA sequence differences in the protein kinase C-beta1 gene (PRKCB1), which encodes both betaI and betaII isoforms, may influence susceptibility to nephropathy. Nine single-nucleotide polymorphisms (SNP) in PRKCB1 were tested for association with diabetic nephropathy in type I diabetes mellitus, by using both case-control and family-study designs. Allele and genotype distributions of two SNP in the promoter (--1504C/T and --546C/G) differed significantly between case patients and control patients (P < 0.05). These associations were particularly strong with diabetes mellitus duration of <24 yr (P = 0.002). The risk of diabetic nephropathy was higher among carriers of the T allele of the --1504C/T SNP, compared with noncarriers (odds ratio, 2.54; 95% confidence interval, 1.39 to 4.62), and among carriers of the G allele of the --546C/G SNP (odds ratio, 2.45; 95% confidence interval, 1.37 to 4.38). Among individuals with diabetes mellitus duration of >/==" BORDER="0">24 yr, these two SNP were not associated with diabetic nephropathy. These positive findings were confirmed by using the family-based transmission disequilibrium test. The T-G haplotype, with both risk alleles, was transmitted more frequently than expected from heterozygous parents to offspring who developed diabetic nephropathy during the first 24 yr of diabetes mellitus. It is concluded that DNA sequence differences in the promoter of PRKCB1 contribute to diabetic nephropathy susceptibility in type I diabetes mellitus.

    Funded by: NIDDK NIH HHS: DK41526, DK53534

    Journal of the American Society of Nephrology : JASN 2003;14;8;2015-24

Literature (245)

Pubmed - other

  • Protein kinase Cbeta modulates ligand-induced cell surface death receptor accumulation: a mechanistic basis for enzastaurin-death ligand synergy.

    Meng XW, Heldebrant MP, Flatten KS, Loegering DA, Dai H, Schneider PA, Gomez TS, Peterson KL, Trushin SA, Hess AD, Smith BD, Karp JE, Billadeau DD and Kaufmann SH

    Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

    Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.

    Funded by: NCI NIH HHS: R01 CA69008

    The Journal of biological chemistry 2010;285;2;888-902

  • Recruitment of PKC-betaII to lipid rafts mediates apoptosis-resistance in chronic lymphocytic leukemia expressing ZAP-70.

    zum Büschenfelde CM, Wagner M, Lutzny G, Oelsner M, Feuerstacke Y, Decker T, Bogner C, Peschel C and Ringshausen I

    3rd Department of Medicine, Hematology and Oncology, Technical University, Munich 81675, Germany.

    ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor to downstream signaling pathways. Its expression in leukemic B-cells derived from a subgroup of patients with chronic lymphocytic leukemia (CLL) is associated with an aggressive course of the disease. However, its implication for the pathogenesis of aggressive CLL is still unclear. In this study, we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor, recruiting protein kinase C-betaII (PKC-betaII) into lipid raft domains. Subsequently, PKC-betaII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein Bim(EL) are putative substrates for PKC-betaII. PKC-betaII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic Bim(EL.) In addition, the phosphorylation of Bim(EL) by PKC-betaII leads to its proteasomal degradation. These changes confer leukemic cells to a more antiapoptotic state with aggressiveness of the disease. Most importantly, these molecular changes can be therapeutically targeted with the small molecule inhibitor Enzastaurin. We provide evidence that this compound is highly active in leukemic cells and augments the cytotoxic effects of standard chemotherapeutic drugs.

    Leukemia 2010;24;1;141-52

  • Cell invasion of Yersinia pseudotuberculosis by invasin and YadA requires protein kinase C, phospholipase C-gamma1 and Akt kinase.

    Uliczka F, Kornprobst T, Eitel J, Schneider D and Dersch P

    Institut für Mikrobiologie, Technische Universität Braunschweig, 38106 Braunschweig, Germany.

    The outer membrane proteins YadA and invasin of Yersinia pseudotuberculosis promote invasion into mammalian cells through beta(1)-integrins and trigger the production of interleukin (IL)-8. FAK, c-Src and the PI3 kinase were previously found to be important for both YadA- and invasin-promoted uptake. Here, we demonstrate that two different downstream effectors of PI3 kinase, Akt and phospholipase Cgamma1 are required for efficient cell invasion. Inhibition of Akt or phospholipase C-gamma (PLC-gamma)1 by pharmaceutical agents as well as reduced expression of the isoforms Akt1 and Akt2, and of PLC-gamma1 by RNA interference decreased entry of YadA- and Inv-expressing bacteria significantly. In addition, we report that the conventional protein kinases C (PKC)alpha and -beta, positioned downstream of PLC-gamma1, are activated upon Inv- or YadA-promoted cell entry. They colocalize with intracellular bacteria and their depletion by siRNA treatment also resulted in a strong reduction of cell entry. In contrast, neither Akt nor PLC-gamma1, and the PKCs are essential for YadA- and Inv-mediated IL-8 synthesis and release. We conclude that YadA and invasin of Y. pseudotuberculosis both trigger similar signal transduction pathways during integrin-mediated phagocytosis into epithelial cells, which lead to the activation of Akt, PLC-gamma1, PKCalpha and -beta downstream of PI3 kinase, separate from the MAPK-dependent pathway that triggers IL-8 production.

    Cellular microbiology 2009;11;12;1782-801

  • Swiprosin-1 is expressed in mast cells and up-regulated through the protein kinase C beta I/eta pathway.

    Thylur RP, Kim YD, Kwon MS, Oh HM, Kwon HK, Kim SH, Im SH, Chun JS, Park ZY and Jun CD

    Department of Life Science, Cell Dynamics Research Center, BioImaging Research Center, and Research Center for Biomolecular Nanotechnology, GIST, Gwangju 500-712, South Korea.

    Swiprosin-1 exhibits the highest expression in CD8(+) T cells and immature B cells and has been thought to play a role in lymphocyte physiology. Here we report that swiprosin-1 is also expressed in mast cells and up-regulated in both in vitro cultured mast cells by phorbol ester and in vivo model tissues of passive cutaneous anaphylaxis and atopic dermatitis. Targeted inhibition of the specific protein kinase C (PKC) isotypes by siRNA revealed that PKC-beta I/eta are involved in the expression of swiprosin-1 in the human mast cell line HMC-1. In contrast, down-regulation of swiprosin-1 by A23187 or ionomycin suggests that calcium-signaling plays a negative role. The ectopic expression of swiprosin-1 augmented PMA/A23187-induced NF-kappaB promoter activity, and resulted in increased expression of cytokines. Moreover, knock-down of swiprosin-1 attenuated PMA/A23187-induced cytokine expression. Collectively, these results suggest that swiprosin-1 is a PKC-beta I/eta-inducible gene and it modulates mast cell activation through NF-kappaB-dependent pathway.

    Journal of cellular biochemistry 2009;108;3;705-15

  • Protein kinases A and C in post-mortem prefrontal cortex from persons with major depression and normal controls.

    Shelton RC, Hal Manier D and Lewis DA

    Department of Psychiatry, Vanderbilt University, Nashville, TN 37212, USA. richard.shelton@vanderbilt.edu

    Major depression (MDD) is a common and potentially life-threatening condition. Widespread neurobiological abnormalities suggest abnormalities in fundamental cellular mechanisms as possible physiological mediators. Cyclic AMP-dependent protein kinase [also known as protein kinase A (PKA)] and protein kinase C (PKC) are important components of intracellular signal transduction cascades that are linked to G-coupled receptors. Previous research using both human peripheral and post-mortem brain tissue specimens suggests that a subset of depressed patients exhibit reduced PKA and PKC activity, which has been associated with reduced levels of specific protein isoforms. Prior research also suggests that specific clinical phenotypes, particularly melancholia and suicide, may be particularly associated with low activity. This study examined PKA and PKC protein levels in human post-mortem brain tissue samples from persons with MDD (n=20) and age- and sex-matched controls (n=20). Specific PKA subunits and PKC isoforms were assessed using Western blot analysis in post-mortem samples from Brodmann area 10, which has been implicated in reinforcement and reward mechanisms. The MDD sample exhibited significantly lower protein expression of PKA regulatory Ialpha (RIalpha), PKA catalytic alpha (Calpha) and Cbeta, PKCbeta1, and PKCepsilon relative to controls. The melancholic subgroup showed low PKA RIalpha and PKA Cbeta, while the portion of the MDD sample who died by suicide had low PKA RIalpha and PKA Calpha. These data continue to support the significance of abnormalities of these two key kinases, and suggest linkages between molecular endophenotypes and specific clinical phenotypes.

    Funded by: NIMH NIH HHS: MH073630, MH084053, P50 MH084053, R01 MH073630

    The international journal of neuropsychopharmacology 2009;12;9;1223-32

  • Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression.

    Lintas C, Sacco R, Garbett K, Mirnics K, Militerni R, Bravaccio C, Curatolo P, Manzi B, Schneider C, Melmed R, Elia M, Pascucci T, Puglisi-Allegra S, Reichelt KL and Persico AM

    Laboratory of Molecular Psychiatry and Neurogenetics, University Campus Bio-Medico, Rome, Italy.

    Protein kinase C enzymes play an important role in signal transduction, regulation of gene expression and control of cell division and differentiation. The fsI and betaII isoenzymes result from the alternative splicing of the PKCbeta gene (PRKCB1), previously found to be associated with autism. We performed a family-based association study in 229 simplex and 5 multiplex families, and a postmortem study of PRKCB1 gene expression in temporocortical gray matter (BA41/42) of 11 autistic patients and controls. PRKCB1 gene haplotypes are significantly associated with autism (P<0.05) and have the autistic endophenotype of enhanced oligopeptiduria (P<0.05). Temporocortical PRKCB1 gene expression was reduced on average by 35 and 31% for the PRKCB1-1 and PRKCB1-2 isoforms (P<0.01 and <0.05, respectively) according to qPCR. Protein amounts measured for the PKCbetaII isoform were similarly decreased by 35% (P=0.05). Decreased gene expression characterized patients carrying the 'normal' PRKCB1 alleles, whereas patients homozygous for the autism-associated alleles displayed mRNA levels comparable to those of controls. Whole genome expression analysis unveiled a partial disruption in the coordinated expression of PKCbeta-driven genes, including several cytokines. These results confirm the association between autism and PRKCB1 gene variants, point toward PKCbeta roles in altered epithelial permeability, demonstrate a significant downregulation of brain PRKCB1 gene expression in autism and suggest that it could represent a compensatory adjustment aimed at limiting an ongoing dysreactive immune process. Altogether, these data underscore potential PKCbeta roles in autism pathogenesis and spur interest in the identification and functional characterization of PRKCB1 gene variants conferring autism vulnerability.

    Funded by: NICHD NIH HHS: P30 HD015052; NIMH NIH HHS: K02 MH070786, R01 MH079299

    Molecular psychiatry 2009;14;7;705-18

  • Protein kinase C-beta II (PKC-beta II) expression in patients with colorectal cancer.

    Spindler KL, Lindebjerg J, Lahn M, Kjaer-Frifeldt S and Jakobsen A

    Danish Colorectal Cancer Group South, Vejle Hospital, 7100, Vejle, Denmark. Karen-lise.garm.spindler@vgs.regionsyddanmark.dk

    Purpose: Current development of targeted agents for the treatment of colorectal cancer include the clinical evaluation of kinase inhibitors, such as enzastaurin, a serine/threonine kinase inhibitor designed to suppress signaling through Protein Kinase C (PKC) and AKT pathways. Little is known about the expression of PKC-beta in colorectal cancer or the prognostic value in colorectal cancer, which was the focus of the present study.

    Methods: PKC-beta II protein expression was examined in 99 primary colorectal adenocarcinomas and 33 corresponding regional lymph node metastases by immunohistochemistry (IHC). The PKC-beta II immunoreactivity was mutually compared and correlated with survival information of all examined patients.

    Results: Immunohistochemical expression of PKC-beta II was detected in 18/99 carcinomas (18.2%). There was no correlation between PKC-beta II staining and traditional clinicopathological parameters. However the median survival was 2.2 years in PKC-beta II expressing tumors compared to 5.4 in PKC-beta II negative tumors (p = 0.25), with a trend for association to poor prognosis.

    Conclusion: We here describe for the first time the immunohistochemical detection of PKC-beta II in patients with colorectal cancer and show a trend associating with poor survival. The role of PKC-beta II staining in colorectal tumors deserves further evaluation.

    International journal of colorectal disease 2009;24;6;641-5

  • PKCbeta is essential for the development of chronic lymphocytic leukemia in the TCL1 transgenic mouse model: validation of PKCbeta as a therapeutic target in chronic lymphocytic leukemia.

    Holler C, Piñón JD, Denk U, Heyder C, Hofbauer S, Greil R and Egle A

    Laboratory for Immunological and Molecular Cancer Research, 3rd Medical Department for Hematology, Oncology, Hemostasiology, Infectious Diseases and Rheumatology, University Hospital Salzburg, Salzburg, Austria.

    The development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signaling via the B-cell receptor (BCR). Protein kinase C beta (PKCbeta) is an essential signaling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKCbeta in the development of murine CLL. TCL1 overexpression did restore the CD5(+) B-cell population that is absent in PKCbeta-deficient mice. However, PKCbeta-deleted TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKCbeta in the establishment of the malignant clone. Moreover, targeting of PKCbeta with the specific inhibitor enzastaurin led to killing of human CLL samples in vitro. We thus propose that PKCbeta may be a relevant target for the treatment of CLL.

    Blood 2009;113;12;2791-4

  • Role of protein kinase C beta2 activation in TNF-alpha-induced human vascular endothelial cell apoptosis.

    Wang F, Liu HM, Irwin MG, Xia ZY, Huang Z, Ouyang J and Xia Z

    Anesthesiology Research Laboratory, Renmin Hospital, Wuhan University, Wuhan, Hubei, China.

    The circulatory inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is increased in pathologic conditions that initiate or exacerbate vascular endothelial injury, such as diabetes. Protein kinase C (PKC) has been shown to play a critical role in TNF-alpha-induced human endothelial cell apoptosis. However, the relative roles played by specific isoforms of PKC in TNF-alpha-induced human endothelial cell apoptosis have not been addressed. We investigated the effects of a selective PKCbeta(2) inhibitor (CGP53353) on TNF-alpha-induced apoptosis in human vascular endothelial cells (cell line ECV304) and on the production of reactive oxygen species and nitric oxide, and compared its effects with rottlerin, a reagent that has been shown to reduce PKCdelta protein levels. Cultured human vascular endothelial cells (ECV304) were treated for 24 h with one of 4 regimes: 40 ng/mL TNF-alpha alone (TNF-alpha), TNF-alpha with 10 micromol/L rottlerin (T+rottlerin), TNF-alpha with 1 micromol/L CGP53353 (T+CGP), or untreated (control). Cell viability was measured by MTT assay, and cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with dramatic increases in production of intracellular hydrogen peroxide (approximately 20 times greater than control) and superoxide (approximately 16 times greater than control), as measured by dichlorofluorescein and dihydroethidium fluorescent staining, respectively. This increase was accompanied by reduced activity of superoxide dismutase and glutathione peroxidase and, subsequently, an increase in the lipid peroxidation product malondialdehyde. CGP53353, but not rottlerin, abolished or attenuated all these changes. We conclude that PKCbeta(2) plays a major role in TNF-alpha-induced human vascular endothelial cell apoptosis.

    Canadian journal of physiology and pharmacology 2009;87;3;221-9

  • The chaperones Hsp90 and Cdc37 mediate the maturation and stabilization of protein kinase C through a conserved PXXP motif in the C-terminal tail.

    Gould CM, Kannan N, Taylor SS and Newton AC

    Pharmacology Department, University of California, San Diego, La Jolla, California 92039-0721, USA.

    The life cycle of protein kinase C (PKC) is tightly controlled by mechanisms that mature the enzyme, sustain the activation-competent enzyme, and degrade the enzyme. Here we show that a conserved PXXP motif (Kannan, N., Haste, N., Taylor, S. S., and Neuwald, A. F. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1272-1277), in the C-terminal tail of AGC (c-AMP-dependent protein kinase/protein kinase G/protein kinase C) kinases, controls the processing phosphorylation of conventional and novel PKC isozymes, a required step in the maturation of the enzyme into a signaling-competent species. Mutation of both Pro-616 and Pro-619 to Ala in the conventional PKC betaII abolishes the phosphorylation and activity of the kinase. Co-immunoprecipitation studies reveal that conventional and novel, but not atypical, PKC isozymes bind the chaperones Hsp90 and Cdc37 through a PXXP-dependent mechanism. Inhibitors of Hsp90 and Cdc37 significantly reduce the rate of processing phosphorylation of PKC. Of the two C-terminal sites processed by phosphorylation, the hydrophobic motif, but not the turn motif, is regulated by Hsp90. Overlay of purified Hsp90 onto a peptide array containing peptides covering the catalytic domain of PKC betaII identified regions surrounding the PXXP segment, but not the PXXP motif itself, as major binding determinants for Hsp90. These Hsp90-binding regions, however, are tethered to the C-terminal tail via a "molecular clamp" formed between the PXXP motif and a conserved Tyr (Tyr-446) in the alphaE-helix. Disruption of the clamp by mutation of the Tyr to Ala recapitulates the phosphorylation defect of mutating the PXXP motif. These data are consistent with a model in which a molecular clamp created by the PXXP motif in the C-terminal tail and determinants in the alphaE-helix of the catalytic domain allows the chaperones Hsp90 and Cdc37 to bind newly synthesized PKC, a required event in the processing of PKC by phosphorylation.

    Funded by: NIDDK NIH HHS: P01 DK54441; NIGMS NIH HHS: 2T32 GM-07752, GM-43154

    The Journal of biological chemistry 2009;284;8;4921-35

  • Protein kinase C isoenzymes differentially regulate the differentiation-dependent expression of adhesion molecules in human epidermal keratinocytes.

    Szegedi A, Páyer E, Czifra G, Tóth BI, Schmidt E, Kovács L, Blumberg PM and Bíró T

    Department of Dermatology, University of Debrecen, Debrecen, Hungary.

    Epidermal expression of adhesion molecules such as desmogleins (Dsg) and cadherins is strongly affected by the differentiation status of keratinocytes. We have previously shown that certain protein kinase C (PKC) isoforms differentially alter the growth and differentiation of human epidermal HaCaT keratinocytes. In this paper, using recombinant overexpression and RNA interference, we define the specific roles of the different PKC isoenzymes in modulation of expression of adhesion molecules in HaCaT keratinocytes. The level of Dsg1, a marker of differentiating keratinocytes, was antagonistically regulated by two Ca-independent 'novel' nPKC isoforms; i.e. it increased by the differentiation-promoting nPKCdelta and decreased by the growth-promoting nPKCepsilon. The expression of Dsg3 (highly expressed in proliferating epidermal layers) was conversely regulated by these isoenzymes, and was also inhibited by the differentiation inducer Ca-dependent 'conventional' cPKCalpha. Finally, the expression of P-cadherin (a marker of proliferating keratinocytes) was regulated by all of the examined PKCs, also in an antagonistic manner (inhibited by cPKCalpha/nPKCdelta and stimulated by cPKCbeta/nPKCepsilon). Collectively, the presented results strongly argue for the marked, differential, and in some instances antagonistic roles of individual Ca-dependent and Ca-independent PKC isoforms in the regulation of expression of adhesion molecules of desmosomes and adherent junctions in human epidermal keratinocytes.

    Experimental dermatology 2009;18;2;122-9

  • Role of PKCbeta in hepatocellular carcinoma cells migration and invasion in vitro: a potential therapeutic target.

    Guo K, Li Y, Kang X, Sun L, Cui J, Gao D and Liu Y

    Liver Cancer Institute, Zhongshan Hospital, Fudan University, No. 180 Fenglin Road, Shanghai, China. guo.kun@zs-hospital.sh.cn

    Considerable interests have recently been focused on mechanism of human hepatocellular carcinoma (HCC) metastasis-the most fundamental characteristics of HCC and the ultimate cause of most HCC mortality, so screening more potential early prognostic marker and therapeutic target is urgent. In this study, we screened genome of three HCC cell lines with consistently increased metastatic potentials and sharing same genetic background, through DNA microarray and found consecutively up-regulated expression of PKCbeta in these cell lines compared to others PKCs, which was reconfirmed by real time RT-PCR and western blot analysis. Moreover, it was found, after efficient silence of PKCbeta by RNAi assay or inhibition of PKCbeta activity by a specific inhibitor LY317615, migration and invasion of HCC cells significantly decreased. In addition, depletion of PKCbeta protein significantly reversed the enhancement of PMA-stimulated HCC migration and invasion ability in vitro. All the data suggest a key role of PKCbeta in HCC motility and PKCbeta may be a potential therapeutic target.

    Clinical & experimental metastasis 2009;26;3;189-95

  • Inhibition of protein kinase Cbeta prevents foam cell formation by reducing scavenger receptor A expression in human macrophages.

    Osto E, Kouroedov A, Mocharla P, Akhmedov A, Besler C, Rohrer L, von Eckardstein A, Iliceto S, Volpe M, Lüscher TF and Cosentino F

    Department of Cardiology, University Hospital and Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich, Switzerland.

    Background: Low-density lipoprotein (LDL) uptake by monocyte-derived macrophages is a crucial step in foam cell formation and early atherosclerotic lesion. Increasing evidence supports the theory that activation of protein kinase Cbeta (PKCbeta) is involved in many mechanisms promoting atherosclerosis. Thus, we investigated whether inhibition of PKCbeta prevents foam cell formation.

    The differentiation of human primary monocytes or the monocytic THP-1 cell line into monocyte-derived macrophages was induced by phorbol 12-myristate 13-acetate (PMA; 0.1 mmol/L), a potent activator of PKC. Incubation of monocyte-derived macrophages with DiI-modified LDL (acetylated LDL and oxidized LDL, 10 mug/mL) led to lipoprotein uptake. Interestingly enough, the nonselective inhibitor of PKCbeta(1) and PKCbeta(2), LY379196 (5x10(-7) to 10(-5) mol/L), blunted LDL uptake in monocyte-derived macrophages as shown by flow cytometry. Specific siRNA-mediated knockdown of PKCbeta exerted a similar effect. Furthermore, PMA alone and in the presence of modified LDL induced scavenger receptor A mRNA and protein expression, which was abolished by LY379196. CGP53353, a selective inhibitor of PKCbeta(2), did not affect LDL uptake, nor did it prevent scavenger receptor A upregulation. Incubation of monocyte-derived macrophages with PMA/LDL increased PKCbeta(1) phosphorylation at the Thr-642 residue, which was blunted by LY379196. However, the expression of CD68, a marker of activated macrophages, was not affected by LY379196. Moreover, LY379196 did not affect lipopolysaccharide-induced CD14 degradation, tumor necrosis factor-alpha release, or superoxide anion production, ruling out any effect of PKCbeta inhibition on innate immunity.

    Conclusions: Nonspecific inhibition of PKCbeta prevents LDL uptake in macrophages. These findings suggest that PKCbeta inhibitors may represent a novel class of antiatherosclerotic drugs.

    Circulation 2008;118;21;2174-82

  • Eukaryotic ribosomes host PKC activity.

    Grosso S, Volta V, Vietri M, Gorrini C, Marchisio PC and Biffo S

    Laboratory of Molecular Histology and Cell Growth, DIBIT, Via Olgettina 58, HSR, 20132 Milano, Italy.

    PKC isoform betaII modulates translation and can be recruited on ribosomes via its scaffold RACK1 (receptor for activated protein kinase C 1), which resides on the 40S ribosomal subunit. However, whether a PKC activity exists on the ribosome is not yet demonstrated. We purified native ribosomes by two different techniques, which avoid stripping of initiation factors and other associated proteins. In both cases, purified ribosomes are able to phosphorylate a specific PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate). MARCKS phosphorylation is switched on by treatment with PKC agonist PMA (Phorbol 12-Myristate 13-Acetate). Consistently, the broad PKC inhibitor BMI (Bisindolyl Maleimide I) abrogates MARCKS phosphorylation. These data show that native ribosomes host active PKC and hence allow the phosphorylation of ribosome-associated substrates like initiation factors and mRNA binding proteins.

    Funded by: Worldwide Cancer Research: 05-0360

    Biochemical and biophysical research communications 2008;376;1;65-9

  • PKCbetaII modulates translation independently from mTOR and through RACK1.

    Grosso S, Volta V, Sala LA, Vietri M, Marchisio PC, Ron D and Biffo S

    Molecular Histology and Cell Growth, HSR, 20132 Milan, Italy.

    RACK1 (receptor for activated C kinase 1) is an abundant scaffolding protein, which binds active PKCbetaII (protein kinase C betaII) increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity to the mRNA exit channel. In the present study we tested the hypothesis that PKCbetaII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. We find that specific inhibition of PKCbetaI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR (mammalian target of rapamycin) targets. These results suggest that PKCbetaII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. Using far-Western analysis, we found that PKCbetaII binds ribosomes in vitro. Co-immunoprecipitation studies indicate that a small but reproducible pool of PKCbetaII is associated with membranes containing ribosomes, suggesting that in vivo PKCbetaII may also physically interact with the ribosomal machinery. Polysomal profiles show that stimulation of PKC results in an increased polysomes/80S ratio, associated with a shift of PKCbetaII to the heavier part of the gradient. A RACK1-derived peptide that inhibits the binding of active PKCbetaII to RACK1 reduces the polysomes/80S ratio and methionine incorporation, suggesting that binding of PKCbetaII to RACK1 is important for PKC-mediated translational control. Finally, down-regulation of RACK1 by siRNA (small interfering RNA) impairs the PKC-mediated increase of translation. Taken together the results of the present study show that PKCbetaII can act as a specific PKC isoform regulating translation, in an mTOR-independent fashion, possibly close to the ribosomal machinery.

    Funded by: Worldwide Cancer Research: 05-0360

    The Biochemical journal 2008;415;1;77-85

  • PKCbetaII augments NF-kappaB-dependent transcription at the CCL11 promoter via p300/CBP-associated factor recruitment and histone H4 acetylation.

    Clarke DL, Sutcliffe A, Deacon K, Bradbury D, Corbett L and Knox AJ

    Division of Respiratory Medicine, University of Nottingham, City Hospital, Nottingham, United Kingdom.

    The transcription factor NF-kappaB plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators, including histone acetyltransferases (HATs) such as CREB-binding protein/p300 and p300/CBP-associated factor (p/CAF). The molecular mechanisms regulating cofactor recruitment are poorly understood. In this study, we describe a novel role for protein kinase C (PKC) betaIotaIota in augmenting NF-kappaB-mediated TNF-alpha-induced transcription of the target gene CCL11 in human airway smooth muscle cells by phosphorylating the HAT p/CAF. Studies using reporters, overexpression strategies, kinase-dead and HAT-defective mutants, and chromatin immunoprecipitation showed that PKCbetaII activation was not involved in NF-kappaB translocation, but facilitated NF-kappaB-mediated CCL11 transcription by colocalizing with and phosphorylating p/CAF, and thereby acetylating histone H4 and promoting p65 association with the CCL11 promoter. The effect was dependent on p/CAF's HAT activity. Furthermore, mouse embryonic fibroblasts from PKCbeta knockout mice showed markedly reduced TNF-alpha-induced CCL11 expression and NF-kappaB reporter activity that was restored on PKCbetaII overexpression, suggesting a critical role for this pathway. These data suggest a novel important biological role for PKCbetaIotaIota in NF-kappaB-mediated CCL11 transcription by p/CAF activation and histone H4 acetylation.

    Funded by: Wellcome Trust

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;5;3503-14

  • No support for association of protein kinase C, beta 1 (PRKCB1) gene promoter polymorphisms c.-1504C>T and c.-546C>G with diabetic nephropathy in Type 1 diabetes.

    Pettigrew KA, McKnight AJ, Martin RJ, Patterson CC, Kilner J, Sadlier D, Maxwell AP, Savage DA and Warren 3/UK GoKinD Study Group

    Diabetic medicine : a journal of the British Diabetic Association 2008;25;9;1127-9

  • Centrosomal PKCbetaII and pericentrin are critical for human prostate cancer growth and angiogenesis.

    Kim J, Choi YL, Vallentin A, Hunrichs BS, Hellerstein MK, Peehl DM and Mochly-Rosen D

    Department of Chemical and Systems Biology, Stanford University, School of Medicine, Stanford, California 94305-5174, USA.

    Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth, but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCbetaII was activated during increased angiogenesis and that PKCbetaII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCbetaII-selective inhibitor, betaIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCbetaII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCbetaII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be up-regulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is up-regulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCbetaII inhibitor such as betaIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.

    Funded by: NCI NIH HHS: CA09151, T32 CA009151; NHLBI NIH HHS: R01 HL076675, R01 HL076675-02, R01 HL076675-03

    Cancer research 2008;68;16;6831-9

  • Apolipoprotein CIII links hyperlipidemia with vascular endothelial cell dysfunction.

    Kawakami A, Osaka M, Tani M, Azuma H, Sacks FM, Shimokado K and Yoshida M

    Department of Geriatrics and Vascular Medicine, Life Science and Bioethics Research Center, Tokyo Medical and Dental University, Tokyo, Japan. kawakami.vasc@tmd.ac.jp

    Background: Apolipoprotein CIII (apoCIII) is a component of some triglyceride-rich very-low-density and low-density lipoprotein and is elevated in dyslipidemia with insulin resistance and the metabolic syndrome. We previously reported that apoCIII directly activates proinflammatory and atherogenic signaling in vascular endothelial cells through protein kinase C-beta (PKCbeta). Because PKCbeta impairs the response of vascular endothelial cells to insulin, we tested the hypothesis that apoCIII affects insulin signaling in vascular endothelial cells and its function in vitro and in vivo.

    ApoCIII inhibited insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), decreasing phosphatidylinositol 3-kinase (PI3K)/Akt activation in human umbilical vein endothelial cells. These effects of apoCIII led to reduced endothelial nitric oxide synthase (eNOS) activation and NO release into the media. ApoCIII activated PKCbeta in human umbilical vein endothelial cells, resulting in IRS-1 dysfunction via serine phosphorylation. ApoCIII also activated mitogen-activated protein kinase through PKCbeta. The impaired insulin signaling was restored by PKCbeta inhibitor or MEK1 inhibitor. ApoCIII-rich very-low-density lipoprotein and apoCIII impaired insulin signaling in the aorta of C57BL/6J mice and in human umbilical vein endothelial cells, which was recovered by PKCbeta inhibitor. They also inhibited endothelium-dependent relaxation of the aortas of C57BL/6J mice. In summary, apoCIII in very-low-density lipoprotein impaired insulin stimulation of NO production by vascular endothelium and induced endothelial dysfunction in vivo. This adverse effect of apoCIII was mediated by its activation of PKCbeta, which inhibits the IRS-1/PI3K/Akt/eNOS pathway.

    Conclusions: Our results suggest that apoCIII is a crucial link between dyslipidemia and insulin resistance in vascular endothelial cells with consequential deleterious effects on their atheroprotective functions.

    Funded by: NIDDK NIH HHS: P30 DK040561

    Circulation 2008;118;7;731-42

  • Counter regulatory effects of PKCbetaII and PKCdelta on coronary endothelial permeability.

    Gaudreault N, Perrin RM, Guo M, Clanton CP, Wu MH and Yuan SY

    Department of Surgery, University of California Davis School of Medicine, 4625 2nd Avenue, Room 3006, Sacramento, CA 95817, USA.

    Objective: The aim of this study was to examine the endothelial distribution and activity of selected PKC isoforms in coronary vessels with respect to their functional impact on endothelial permeability under the experimental conditions relevant to diabetes.

    En face immunohistochemistry demonstrated a significant increase of PKC(betaII) and decrease of PKCdelta expression in coronary arterial endothelium of Zucker diabetic rats. To test whether changes in PKC expression alter endothelial barrier properties, we measured the transcellular electric resistance in human coronary microvascular endothelial monolayers and found that either PKC(betaII) overexpression or PKCdelta inhibition disrupted the cell-cell adhesive barrier. Three-dimensional fluorescence microscopy revealed that hyperpermeability was caused by altered PKC activity in association with distinct translocation of PKC(betaII) to the cell-cell junction and PKCdelta localization to the cytosol. Further analyses in fractionated endothelial lysates confirmed the differential redistribution of these isozymes. Additionally, FRET analysis of PKC subcellular dynamics demonstrated a high PKC(betaII) activity at the cell surface and junction, whereas PKCdelta activity is concentrated in intracellular membrane organelles.

    Conclusions: Taken together, these data suggest that PKC(betaII) and PKCdelta counter-regulate coronary endothelial barrier properties by targeting distinctive subcellular sites. Imbalanced PKC(betaII)/PKCdelta expression and activity may contribute to endothelial hyperpermeability and coronary dysfunction in diabetes.

    Funded by: NHLBI NIH HHS: HL061507, HL070752, HL073324, HL084542, R01 HL061507, R01 HL070752, R01 HL070752-06, R01 HL073324, R01 HL084542, R01 HL084542-03

    Arteriosclerosis, thrombosis, and vascular biology 2008;28;8;1527-33

  • PKC delta in preeclamptic placentas promotes Bax dissociation from 14-3-3 zeta through 14-3-3 zeta phosphorylation.

    Park JK, Kang MY, Kim YH, Jo HC, Shin JK, Choi WJ, Lee SA, Lee JH, Choi WS and Paik WY

    Department of Obstetrics and Gynecology, College of Medicine, Gyeongsang National University, 90 Chilam-dong, JinJu 660-702, Republic of Korea.

    Objective: We investigated placental apoptosis and the expression of and interactions between 14-3-3 and Bcl-2 family proteins during preeclampsia. In addition, we explored the mechanism of Bax dissociation from 14-3-3, hypothesizing that PKC-mediated phosphorylation of 14-3-3 results in dissociation of Bax from 14-3-3 proteins, and leads to apoptosis.

    Methods: Placental samples from 10 women with preeclampsia and 10 normotensive control patients were analyzed using M30-specific immunohistochemistry to assess placental apoptosis. Biochemical markers of cellular apoptosis, such as cleaved caspase-3, Bax, Bcl-2, 14-3-3, and PKC were followed by Western blotting. Interaction of 14-3-3 proteins with Bax and with PKC was assessed by immunoprecipitation.

    Results: M30-positive cells were widespread in the preeclamptic placentas. The levels of cleaved caspase-3, Bax, 14-3-3 zeta, phospho-(Ser)-14-3-3, and PKC delta were significantly higher in the preeclamptic placentas than in normal placentas. Preeclampsia was also associated with weaker interactions between 14-3-3 zeta and Bax and stronger interactions between 14-3-3 zeta and PKC delta.

    Conclusion: Our results suggest that PKC delta in preeclamptic placentas promotes Bax dissociation from 14-3-3 zeta through the phosphorylation of 14-3-3 zeta. This finding may at least in part explain the apoptosis-inducing activity of PKC delta, revealing the important role of PKC delta in the development of apoptosis-related diseases such as preeclampsia.

    Placenta 2008;29;7;584-92

  • Differential modulation of unapposed connexin 43 hemichannel electrical conductance by protein kinase C isoforms.

    Hawat G and Baroudi G

    Centre de Biomédecine, Hôpital du Sacré-Coeur de Montréal, Montréal, Québec, Canada.

    Opening of unapposed connexin 43 hemichannels (Cx43Hc) in the plasma membrane results in altered ionic homeostasis leading to cell damage. Although it is generally acknowledged that Cx43Hc function is regulated by protein kinase C (PKC), information regarding the functional role of PKC in the modulation of Cx43Hc electrical conductance is lacking. In this work, we used the patch-clamp technique to study the effect of phorbol 12-myristate 13-acetate (PMA), a general PKC activator, on the electrical conductance of exogenous Cx43Hc expressed in tsA201 cells. Subsequently, a matrix of synthetic PKC isoform-specific inhibitor peptides was used to dissect the functional role of individual PKC isoforms in Cx43Hc regulation. Superfusion with 10 nM PMA abolished Cx43Hc currents by 74%, an effect that was prevented by pretreatment with a general PKC inhibitor, GF109203X. It is interesting to note that intracellular diffusion of epsilon V1-2 (0.1 microM), an epsilon PKC-specific inhibitor peptide, completely antagonized PMA-induced current inhibition. Cell dialysis with either beta II- or delta PKC inhibitor peptides partially decreased PMA effect. Neither alpha- nor beta I PKC inhibition altered PMA-induced current reduction. This study shows for the first time that Cx43Hc electrical conductance is inhibited after PKC activation. Moreover, this inhibition is predominantly mediated by the "novel" epsilon PKC isoform, whereas partial inhibition may be provided by the "conventional" beta II PKC as well as the "novel" delta PKC isoforms.

    Pflugers Archiv : European journal of physiology 2008;456;3;519-27

  • Identification of ubiquitin ligase activity of RBCK1 and its inhibition by splice variant RBCK2 and protein kinase Cbeta.

    Tatematsu K, Yoshimoto N, Okajima T, Tanizawa K and Kuroda S

    Department of Structural Molecular Biology, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. kenji44@sanken.osaka-u.ac.jp

    We previously identified a RING-IBR protein, RBCK1, as a protein kinase C (PKC) beta- and zeta-interacting protein, and its splice variant, RBCK2, lacking the C-terminal half including the RING-IBR domain. RBCK1 has been shown to function as a transcriptional activator whose nuclear translocation is prevented by interaction with the cytoplasmic RBCK2. We here demonstrate that RBCK1, like many other RING proteins, also possesses a ubiquitin ligase (E3) activity and that its E3 activity is inhibited by interaction with RBCK2. Moreover, RBCK1 has been found to undergo efficient phosphorylation by PKCbeta. The phosphorylated RBCK1 shows no self-ubiquitination activity in vitro. Overexpression of PKCbeta leads to significant increases in the amounts of intracellular RBCK1, presumably suppressing the proteasomal degradation of RBCK1 through self-ubiquitination, whereas coexpression with PKCalpha, PKCepsilon, and PKCzeta shows no or little effect on the intracellular amount of RBCK1. Taken together, the E3 activity of RBCK1 is controlled by two distinct manners, interaction with RBCK2 and phosphorylation by PKCbeta. It is possible that other RING proteins, such as Parkin, BRCA1, and RNF8, having the E3 activity, are also down-regulated by interaction with their RING-lacking splice variants and/or phosphorylation by protein kinases.

    The Journal of biological chemistry 2008;283;17;11575-85

  • Increased renal gene transcription of protein kinase C-beta in human diabetic nephropathy: relationship to long-term glycaemic control.

    Langham RG, Kelly DJ, Gow RM, Zhang Y, Cox AJ, Qi W, Thai K, Pollock CA, Christensen PK, Parving HH and Gilbert RE

    University of Melbourne Department of Medicine, St Vincent's Hospital, Fitzroy, VIC, Australia. rlangham@medstv.unimelb.edu.au

    Activation of protein kinase C (PKC) isoforms has been implicated as a central mediator in the pathogenesis of diabetic nephropathy. Although high glucose levels stimulate catalytic activity of PKC, the effects of high glucose levels on the expression of genes encoding PKC isoforms are unknown. We sought to determine whether in addition to activation, diabetes may lead to increased transcription of two PKC isoforms that have been implicated in the pathogenesis of diabetic nephropathy, PKC-alpha and PKC-beta.

    Methods: Recent advances in molecular biological techniques now permit quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed.

    Results: Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies of diabetic patients relative to control (p<0.001). No increase in PRKC-alpha expression was seen. In addition, a correlation between renal PRKC-beta mRNA and HbA(1c) was observed in diabetic patients (r=0.63, p<0.05). There was co-localisation of PKC-beta and phospho-PKC-beta predominantly to proximal tubules. A 60% increase in PRKC-beta mRNA and peptide in cultured human proximal tubular epithelial cells exposed to high glucose (p<0.05) was seen in vitro.

    PKC-beta is upregulated at the gene expression level in human diabetic nephropathy. PRKC-beta mRNA correlates closely with serum HbA(1c), possibly partially explaining the relationship between glycaemic control and progression of diabetic nephropathy. Archival human tissue provides a valuable resource for molecular analyses.

    Diabetologia 2008;51;4;668-74

  • Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism.

    Seth A, Yan F, Polk DB and Rao RK

    Dept. of Physiology, Univ. of Tennessee Health Science Center, Memphis, TN 38163, USA.

    Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and beta-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCbetaI and PKCepsilon. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

    Funded by: NIAAA NIH HHS: AA12307, R01 AA012307; NIDDK NIH HHS: DK065788, DK54993, DK55532, R01 DK054993, R01 DK054993-05, R01 DK055532, R01 DK056008, R01 DK056008-09, R56 DK055532

    American journal of physiology. Gastrointestinal and liver physiology 2008;294;4;G1060-9

  • Involvement of protein kinase C beta-extracellular signal-regulating kinase 1/2/p38 mitogen-activated protein kinase-heat shock protein 27 activation in hepatocellular carcinoma cell motility and invasion.

    Guo K, Liu Y, Zhou H, Dai Z, Zhang J, Sun R, Chen J, Sun Q, Lu W, Kang X and Chen P

    Liver Cancer Institute, Zhongshan Hospital, Fudan University, No. 180 Fenglin Road, Shanghai 200032, China.

    To understand the molecular mechanism that underlies the role of various prominent signal pathways in hepatocellular carcinoma (HCC) metastasis, a human signal transduction oligonucleotide microarray analysis was carried out in cultured HCC cell models with increasing spontaneous metastatic potential (MHCC97L, MHCC97H, and HCCLM6). The results revealed that the mitogen-activated protein kinase (MAPK) pathway is the prominently upregulated pathway in HCC metastasis. Further study showed that basal phosphorylated levels of extracellular signal-regulating kinase (ERK)(1/2) and p38 MAPK consecutively increased from MHCC97L to MHCC97H to HCCLM6 cells, but not c-Jun N-terminal kinase. The phosphorylation of ERK(1/2) and p38 MAPK was regulated by upregulated protein kinase C beta (PKC beta) in HCC cells through the integrated use of PKC beta RNA interference, the PKC beta specific inhibitor enzastaurin and a PKC activator phorbol-12-myristate-13-acetate. Heat shock protein 27 (HSP27) was also verified as a downstream common activated protein of PKC beta-ERK(1/2) and PKC beta-p38 MAPK. In vitro migration and invasion assay further showed that the depletion of PKC beta or inhibition of PKC beta activation effectively decreased HCC cell motility and invasion. Moreover, the motility and invasion of phorbol-12-myristate-13-acetate-stimulated PKC beta-mediated HCC cells was significantly negated by an ERK inhibitor, 1.4-diamino-2.3-dicyano-1.4-bis[2-aminophenylthio] butadiene, or a p38 MAPK inhibitor, 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole. It also showed that HSP27 is critical in PKC beta-mediated HCC cell motility and invasion. Taken together, this study reveals the important role of this PKC beta-ERK(1/2)/p38MAPK-HSP27 pathway, which was verified for the first time, in modulating HCC cell motility and invasion.

    Cancer science 2008;99;3;486-96

  • S-Phase-specific activation of PKC alpha induces senescence in non-small cell lung cancer cells.

    Oliva JL, Caino MC, Senderowicz AM and Kazanietz MG

    Department of Pharmacology and Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA. jloliva@isciii.es

    Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.

    Funded by: NCI NIH HHS: CA-89202, CA-92537

    The Journal of biological chemistry 2008;283;9;5466-76

  • Role of phospholipase Cgamma-induced activation of protein kinase Cepsilon (PKCepsilon) and PKCbetaI in epidermal growth factor-mediated protection of tight junctions from acetaldehyde in Caco-2 cell monolayers.

    Suzuki T, Seth A and Rao R

    Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.

    Epidermal growth factor (EGF) protects the intestinal epithelial tight junctions from acetaldehyde-induced insult. The role of phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) isoforms in the mechanism of EGF-mediated protection of tight junction from acetaldehyde was evaluated in Caco-2 cell monolayers. EGF-mediated prevention of acetaldehyde-induced decrease in transepithelial electrical resistance and an increase in inulin permeability, and subcellular redistribution of occludin and ZO-1 was attenuated by reduced expression of PLCgamma1 by short hairpin RNA. EGF induced a rapid activation of PLCgamma1 and PLC-dependent membrane translocation of PKCepsilon and PKCbetaI. Inhibition of PKC activity or selective interference of membrane translocation of PKCepsilon and PKCbetaI by RACK interference peptides attenuated EGF-mediated prevention of acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. BAPTA-AM and thapsigargin blocked EGF-induced membrane translocation of PKCbetaI and attenuated EGF-mediated prevention of acetaldehyde-induced disruption of tight junctions. EGF-induced translocation of PKCepsilon and PKCbetaI was associated with organization of F-actin near the perijunctional region. This study shows that PLCgamma-mediated activation of PKCepsilon and PKCbetaI and intracellular calcium is involved in EGF-mediated protection of tight junctions from acetaldehyde-induced insult.

    Funded by: NIAAA NIH HHS: R01-AA12307; NIDDK NIH HHS: R01-DK55532

    The Journal of biological chemistry 2008;283;6;3574-83

  • The context of HLA-DR/CD18 complex in the plasma membrane governs HLA-DR-derived signals in activated monocytes.

    Doisne JM, Castaigne JG, Deruyffelaere C, Dieu-Nosjean MC, Chamot C, Alcaide-Loridan C, Charron D and Al-Daccak R

    INSERM U662, Réponses Immunes: Régulation et Développement, Institut Universitaire d'Hématologie, Université Paris 7, Hôpital St. Louis, 1 Av Claude Vellefaux, 75010 Paris, France.

    HLA-DR-derived signals in activated monocytes mediate both pro-inflammatory cytokine production and caspase-independent death, and have been postulated to play a role in inflammation and in its resolution, respectively. Herein, using the monocytic/macrophagic human cell line THP-1 primed with IFNgamma (IFNgamma-primed THP-1), we investigated how HLA-DR may integrate both signals. Our inhibition studies demonstrated that if cell death is dependent on PKCbeta activation, the induction of TNFalpha gene expression relies on PTK activation, in particular the Src family of kinases, but both cell responses implicate the beta2-integrin CD18. Accordingly, sequential immunoprecipitation experiments demonstrated that following engagement of HLA-DR on IFNgamma-primed THP-1 cells, the HLA-DR/CD18 complex physically associates with PKCbeta and with PTK. Pharmacological disruption of lipid rafts microdomains abolished the assembly of HLA-DR/CD18/PTK signaling complex, HLA-DR-mediated tyrosine activation, and the PTK-dependent TNFalpha expression in IFNgamma-primed THP-1 cells. In contrast, HLA-DR/CD18/PKCbeta complex was still formed and able to mediate cell death after cholesterol depletion of these cells. These results indicate that while the integrity of lipid rafts is necessary for the transduction of cytokine gene expression through the HLA-DR/CD18 complex, it is not necessary for the induction of the HLA-DR/CD18-dependent cell death. Thus, our study provides experimental evidence indicating the compartmentalization of HLA-DR/CD18 complex within or outside lipid rafts as a mechanism through which HLA-DR can integrate both PTK and PKCbeta signals leading to activation and death, respectively, of activated monocytes. This might provide new insights into how MHC class II signaling may regulate inflammatory response.

    Molecular immunology 2008;45;3;709-18

  • Foreign body-type multinucleated giant cell formation requires protein kinase C beta, delta, and zeta.

    McNally AK, Macewan SR and Anderson JM

    Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA. amy.mcnally@case.edu

    Multinucleated giant cells are a classic cellular feature of chronic inflammation, although the mechanism of macrophage fusion leading to their formation is not well understood. Here, we investigate the participation of protein kinase C (PKC) in the interleukin (IL)-4-induced fusion of human monocyte-derived macrophages and foreign body giant cell (FBGC) formation in vitro. The PKC inhibitors H-7 and calphostin C attenuated macrophage fusion, whereas H-8, which is more selective for PKA and PKG, did not. Macrophage fusion was also prevented by the phospholipase C inhibitor, Et-18-OCH(3), the PKC isoform inhibitors GO6983 or rottlerin and by peptide inhibitors for PKC (20-28), PKCbeta, or PKCzeta but not by HBDDE or peptide inhibitors for PKCvarepsilon or PKA. In cultures of fusing macrophages/FBGC, we detected only PKCalpha, beta, delta, and zeta by immunoprecipitation and immunoblotting, and we also observed strong expression of these isoforms by immunocytochemistry. Our collective results suggest that the gamma, epsilon, eta, mu, theta, or iota PKC isoforms are not required in the mechanism of IL-4-induced macrophage fusion; whether PKCalpha is required is unclear. However, new evidence is provided that FBGC formation is supported by PKCbeta, PKCdelta, and PKCzeta in combined diacylglycerol-dependent (PKCbeta and PKCdelta) and -independent (PKCzeta) signaling pathways.

    Funded by: NIBIB NIH HHS: EB000275, EB000282, R01 EB000275, R01 EB000275-22, R01 EB000282, R01 EB000282-12, R37 EB000275, R56 EB000282

    Experimental and molecular pathology 2008;84;1;37-45

  • Identification of a functional protein kinase Cbeta promoter polymorphism in humans related to insulin resistance.

    Osterhoff MA, Heuer S, Pfeiffer M, Tasic J, Kaiser S, Isken F, Spranger J, Weickert MO, Möhlig M and Pfeiffer AF

    German Institute of Human Nutrition, Potsdam-Rehbruecke (DIfE), Department of Clinical Nutrition (KLE), Arthur-Scheunert-Allee 155, D-14558 Nuthetal, Germany. martino@dife.de

    Protein kinase Cbeta (PKCbeta) is known to inhibit insulin production in beta-cells and to support insulin action in skeletal muscle. We therefore searched for functional polymorphisms among already known genetic variants in the PKCbeta promoter and investigated their relation to glucose metabolism in humans. We found that the gene variant in the PKCbeta promoter at position -546 significantly reduced promoter activity in functional assays (P<0.05). Human subjects carrying this variant had a 3.5-fold decrease in PKCbeta2-protein expression in their thrombocytes (P=0.006). Additionally, we tested whether this variant affects parameters of glucose metabolism using 1012 humans included into the MeSyBePo study (Metabolic Syndrome Berlin Potsdam). The -546 variant was highly significant associated with increased homeostasis model assessment for insulin resistance (HOMA-IR, P=0.009) in the cohort. This association was accompanied by significantly increased fasting insulin concentrations in carriers of the homozygous polymorphism (P=0.021). Our results suggest that the -546 polymorphism in the PKCbeta promoter reduces promoter activity, which leads to a decreased expression of PKCbeta2 and subsequently is associated with decreased peripheral insulin-dependent glucose uptake.

    Molecular genetics and metabolism 2008;93;2;210-5

  • Protein kinase C-betaII represses hepatocyte growth factor-induced invasion by preventing the association of adapter protein Gab1 and phosphatidylinositol 3-kinase in melanoma cells.

    Oka M, Kikkawa U and Nishigori C

    Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan. oka@med.kobe-u.ac.jp

    The hepatocyte growth factor (HGF) signaling pathway was examined in human normal melanocytes and three malignant melanoma cell lines. HGF-induced activation of c-Met, its receptor-tyrosine kinase, was observed in both melanocytes and melanoma cells, whereas phosphatidylinositol 3-kinase (PI3K), a downstream target of c-Met, was not activated in the melanocytes but enhanced in the melanoma cell lines. The electrophoretic mobility of Gab1, the scaffolding adapter protein that couples activated c-Met and PI3K, was slower in the melanocytes than that in the melanoma cells, and the mobility shifted to that of the melanoma cells after treatment with alkaline phosphatase, indicating that Gab1 is highly phosphorylated on serine and threonine in the melanocytes. Introduction of protein kinase C (PKC)-betaII into the melanoma cells, which is expressed in melanocytes but absent in melanoma cells, resulted in serine and threonine phosphorylation of Gab1 and also prevented tyrosine phosphorylation of Gab1 and its association with PI3K. Furthermore, the introduction of PKC-betaII suppressed HGF-induced activation of PI3K, and attenuated the in vitro invasion activity of the melanoma cells. These results indicate that the HGF signaling process from Gab1 to PI3K is negatively regulated by PKC-betaII, and its loss is critical for melanoma cells to gain invasive potential.

    The Journal of investigative dermatology 2008;128;1;188-95

  • Protein kinase C beta overexpression induces fibrotic effects in human proximal tubular epithelial cells.

    Slattery C, Ryan MP and McMorrow T

    School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia.

    Cyclosporine A (CsA) significantly improves the success of organ transplantation, however renal fibrosis, characterised by severe tubulointerstitial fibrosis is a complication of CsA therapy. Previously we have reported the involvement of PKC-beta isoforms in a model of CsA-induced tubulointerstitial fibrosis and we have now further elucidated this role. Treatment of human proximal tubular epithelial cells with CsA resulted in increased fibronectin production which coincided with increased PKC activity. To delineate the respective contributions of the two PKC-beta isoforms in fibrotic events, we overexpressed PKC-betaI, -betaII, or both in combination. Overexpression of the two PKC-beta isoforms induced morphological alterations, secretion of the profibrotic cytokine TGF-beta1, and fibronectin release from proximal tubular cells however PKC-betaII induced more significant effects in all parameters examined. Inhibition of PKC-beta completely abrogated the CsA-induced increase in fibronectin secretion demonstrating a direct antifibrotic effect of PKC-beta inhibition. Further studies also identified a role for the p44/42 mitogen activated kinase signalling pathway in CsA-induced fibrotic effects downstream of PKC-beta. Overall, these findings demonstrate a central role for PKC-beta, and particularly PKC-betaII in the development of tubulointerstitial fibrosis and suggest that PKC-beta may be a viable therapeutic target in CsA nephropathy.

    The international journal of biochemistry & cell biology 2008;40;10;2218-29

  • Protein expression and cellular localization in two prognostic subgroups of diffuse large B-cell lymphoma: higher expression of ZAP70 and PKC-beta II in the non-germinal center group and poor survival in patients deficient in nuclear PTEN.

    Fridberg M, Servin A, Anagnostaki L, Linderoth J, Berglund M, Söderberg O, Enblad G, Rosén A, Mustelin T, Jerkeman M, Persson JL and Wingren AG

    Department of Tumor Biology, Lund University, Malmö University Hospital, Malmö, Sweden.

    Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) show varying responses to conventional therapy, and this might be contributed to the differentiation stage of the tumor B-cells. The aim of the current study was to evaluate a panel of kinases (ZAP70, PKC-beta I and II and phosphorylated PKB/Akt) and phosphatases (PTEN, SHP1 and SHP2) known to be frequently deregulated in lymphoid malignancies. De novo DLBCL cases were divided into two subgroups, the germinal center (GC) group (14/28) and the non-germinal center (non-GC) or activated B-cell (ABC) group (14/28). ZAP70 and PKC-beta II were expressed in a significantly higher percentage of tumor cells in the clinically more aggressive non-GC group compared with the prognostically favourable GC group. Also, the subcellular localization of PKC-beta I and II differed in DLBCL cells, with the PKC-beta I isoform being expressed in both the cytoplasm and nucleus, while PKC-beta II was found exclusively in the cytoplasm. Loss of nuclear PTEN correlated with poor survival in cases from both subgroups. In addition, five cell lines of DLBCL origin were analyzed for protein expression and for mRNA levels of PTEN and SHP1. For the first time, we show that ZAP70 is expressed in a higher percentage of tumor cells in the aggressive non-GC subgroup of DLBCL and that PKC-beta I and II are differently distributed in the two prognostic subgroups of de novo DLBCL.

    Leukemia & lymphoma 2007;48;11;2221-32

  • Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation.

    Kuyumcu-Martinez NM, Wang GS and Cooper TA

    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.

    The genetic basis of myotonic dystrophy type 1 (DM1) is a CTG expansion in the 3' untranslated region (UTR) of DMPK. The pathogenic mechanism involves an RNA gain of function in which the repeat-containing transcripts accumulate in nuclei and alter the functions of RNA-binding proteins such as CUG-binding protein 1 (CUGBP1). CUGBP1 levels are increased in DM1 myoblasts, heart, and skeletal muscle tissues and in some DM1 mouse models. However, the molecular mechanisms for increased CUGBP1 in DM1 are unclear. Here, we demonstrate that expression of DMPK-CUG-repeat RNA results in hyperphosphorylation and stabilization of CUGBP1. CUGBP1 is hyperphosphorylated in DM1 tissues, cells, and a DM1 mouse model. Activation of PKC is required for CUGBP1 hyperphosphorylation in DM1 cells, and PKCalpha and betaII directly phosphorylate CUGBP1 in vitro. These results indicate that inappropriate activation of the PKC pathway contributes to the pathogenic effects of a noncoding RNA.

    Funded by: NHLBI NIH HHS: R01 HL045565, R01 HL045565-17, R01HL45565; NIAMS NIH HHS: 1F32AR052630-01, F32 AR052630, R01 AR045653, R01 AR045653-09, R01AR45653

    Molecular cell 2007;28;1;68-78

  • The essentiality of PKCalpha and PKCbetaI translocation for CD14+monocyte differentiation towards macrophages and dendritic cells, respectively.

    Lin YF, Lee HM, Leu SJ and Tsai YH

    Graduate Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan.

    Human peripheral CD14(+)monocytes have been known to differentiate into monocyte-derived macrophages (MDMs) or dendritic cells (MoDCs) upon suitable stimulation. However, the key intracellular molecule(s) associated with their differentiation towards specific cell types was(were) not fully understood. This study was designated to determine the association of PKC isoenzymes with the differentiation of CD14(+)monocytes into MDMs or MoDCs. Purified human peripheral CD14(+)monocytes were cultured with GM-CSF, or GM-CSF plus IL-4 for 7 days to induce cell differentiation. The phenotypic changes were analyzed by Flow-Cytometry using various specific antibodies to cell type-specific surface markers. The immunological functions of these differentiated cells were determined by measuring the amounts of TNF-alpha secretion for MDMs, and the capacities of antigen-capturing and bacterial phagocytosis for MoDCs. The translocations of PKC isoenzymes in these cells from cytosol to plasma membrane were examined by Western Blot analysis and Confocal Microscopic observation. The treatment of CD14(+)monocytes with either GM-CSF or PMA elicited PKCalpha translocation and consequently induced their differentiation into MDMs. The inclusion of PKCalpha/beta(I) specific inhibitor, Go6976, greatly inhibited the GM-CSF-induced PKCalpha translocation and dose-dependently reduced the GM-CSF- induced MDM differentiation. On the other hand, the simultaneous pretreatment of CD14(+)monocytes with Go6976 and PKCbeta-specific inhibitor predominantly suppressed the GM-CSF/IL-4-induced generation of MoDCs. Further study demonstrated that GM-CSF/IL-4 selectively induced the translocation of PKCbeta(I), not PKCalpha or PKCbeta(II), in CD14(+)monocytes. In conclusion, the cell fate commitment of CD14(+)monocytes towards MDMs or MoDCs appears to be steered by the selective activation of PKCalpha or PKCbeta(I), respectively.

    Journal of cellular biochemistry 2007;102;2;429-41

  • Parathyroid hormone stimulates the endothelial nitric oxide synthase through protein kinase A and C pathways.

    Rashid G, Bernheim J, Green J and Benchetrit S

    Department of Nephrology and Hypertension, Meir Medical Center, Tchernichovsky 59, Kfar-Saba 44281, Israel. gloriar@clalit.org.il

    Background: Parathyroid hormone (PTH), the major systemic calcium regulating hormone has been implicated in the development of hypertension and the occurrence of uraemic vascular changes. As nitric oxide synthase (NOS) is involved in the production of nitric oxide, and acute PTH effect is characterized by vasodilation, the effect of PTH on the endothelial NOS (eNOS) system was measured in cultured human umbilical cord vein endothelial cells (HUVEC) and the pathways possibly involved were studied.

    Methods: The presence of the PTH receptor-1 (PTHR1) on the HUVEC membrane was examined by RT-PCR, immunocytochemistry and western blot. HUVEC were stimulated with 10(-12) to 10(-10) mol/l PTH. The eNOS mRNA expression was established by RT-PCR and the eNOS protein levels were determined by western blot. The eNOS activity was measured by the conversion of [(14)C]arginine to [(14)C]citrulline.

    Results: PTHR1 has been found to be expressed in HUVEC and its expression is depressed by increasing concentrations of PTH. PTH induced a significant increase in eNOS mRNA (10(-11) mol/l: 1.87 +/- 0.16, P = 0.012; 10(-10) mol/l: 1.96 +/- 0.28, P = 0.007, fold of control), and protein expression. The eNOS activity was also significantly stimulated (10(-11) mol/l: 1139 +/- 203; 10(-10) mol/l: 1323 +/- 216 vs control: 621 +/- 154 cpm/150 mug protein, P < 0.01). The addition of calphostin C (PKC inhibitor) or Rp-cAMP (PKA inhibitor) reduced the eNOS mRNA, protein expression and activity of PTH-stimulated HUVEC. The combined treatment of calphostin C and Rp-cAMP abolished the eNOS protein expression and activity.

    Conclusion: PTH induces an increased activity of the eNOS system; probably both PKA and PKC pathways are involved in this activation. Such data may explain the vasodilation observed after acute treatment with PTH.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2007;22;10;2831-7

  • Differential protein kinase C isoform abundance in ascending aortic aneurysms from patients with bicuspid versus tricuspid aortic valves.

    Jones JA, Stroud RE, Kaplan BS, Leone AM, Bavaria JE, Gorman JH, Gorman RC and Ikonomidis JS

    Division of Cardiothoracic Surgery, Department of Surgery, Medical University of South Carolina, Charleston, SC 29425, USA.

    Background: It is recognized that different events contribute to the initiation of ascending thoracic aortic aneurysms (ATAAs) in patients with bicuspid aortic valves (BAV) versus patients with tricuspid aortic valves (TAV), but the molecular signaling pathways driving aneurysm formation remain unclear. Protein kinase C (PKC) is a superfamily of kinases which differentially mediate signaling events that lead to altered gene expression and cellular function, and may regulate downstream mediators of vascular remodeling. The present study tested the hypothesis that ATAA development in patients with BAV versus TAV proceeds by independent signaling pathways involving differential PKC signaling.

    ATAA samples were collected from BAV (n=57) and TAV (n=55) patients and assessed for 10 different PKC isoforms by immunoblotting. Results were expressed as a percent change in abundance (mean+/-SEM) from a nonaneurysmal control group (100%, n=21). Correlation analysis was performed, and relationships between PKC and matrix metalloproteinase abundance were reported. In the BAV group, classic and novel PKC isoforms (PKC-alpha, betaI, gamma, epsilon, theta) were increased, whereas PKC-eta and atypical PKC-zeta were decreased. In the TAV group, classic and novel isoforms were decreased and atypical PKC-zeta was elevated. Positive correlations between PKC and matrix metalloproteinase abundance were identified.

    Conclusions: Differential PKC isoform abundance was observed in ATAA samples from patients with BAV versus TAV, suggesting independent molecular signaling pathways may be operative. Induction of independent transcriptional programs may result and may provide a mechanistic foundation for developing selective diagnostic/therapeutic strategies for patients with ATAAs secondary to BAV or TAV.

    Funded by: NHLBI NIH HHS: R01 HL075488-04

    Circulation 2007;116;11 Suppl;I144-9

  • Human biliverdin reductase, a previously unknown activator of protein kinase C betaII.

    Maines MD, Miralem T, Lerner-Marmarosh N, Shen J and Gibbs PE

    Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14624, USA. mahin_maines@urmc.rochester.edu

    Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC betaII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC betaII underscores its potential function in propagation of signals relayed through PKCs.

    Funded by: NIEHS NIH HHS: ES004066, ES12187

    The Journal of biological chemistry 2007;282;11;8110-22

  • Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

    Wissing J, Jänsch L, Nimtz M, Dieterich G, Hornberger R, Kéri G, Wehland J and Daub H

    Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany.

    Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.

    Molecular & cellular proteomics : MCP 2007;6;3;537-47

  • Role of atypical protein kinase C isozymes and NF-kappaB in IL-1beta-induced expression of cyclooxygenase-2 in human myometrial smooth muscle cells.

    Duggan SV, Lindstrom T, Iglesias T, Bennett PR, Mann GE and Bartlett SR

    Cardiovascular Division, School of Biomedical and Health Sciences, King's College London, Guy's Campus, London, United Kingdom.

    Increased myometrial expression of cyclooxygenase-2 (Cox-2) at term results from elevated local levels of inflammatory cytokines, and its inhibition provides a potential route for intervention in human pre-term labor. We have identified a role for atypical protein kinase C (PKC) isozymes in IL-1beta-induced Cox-2 expression in human myometrial smooth muscle cells (HMSMC). The PKC inhibitor GF109203X (10 microM) inhibited IL-1beta-induced Cox-2 protein and RNA expression, which were also reduced by MAPK and nuclear factor kappaB (NF-kappaB) inhibitors. GF109203X did not affect MAPK activities, and neither did it replicate the effect of p38 MAPK inhibition on Cox-2 mRNA stability, suggesting that PKC operates through an independent mechanism. The effect of GF109203X remained intact after depletion of conventional and novel PKC isozymes by phorbol ester pre-treatment. In contrast LY379196 (10 microM), which at micromolar concentrations inhibits all but atypical PKCs, did not affect Cox-2 expression. A peptide corresponding to the pseudosubstrate sequence of atypical PKCs blocked Cox-2 protein expression, whereas the sequence from conventional PKCs was ineffective. GF109203X did not affect NF-kappaB binding to nuclear proteins, but strongly reduced NF-kappaB-dependent transcription in luciferase reporter assays. Our findings indicate that IL-1beta-induced Cox-2 expression in HMSMC in culture requires p38-MAPK-mediated mRNA stabilization and an independent activation of Cox-2 transcription which is dependent on the action of atypical PKCs, probably through direct stimulation of the transactivating activity of NF-kappaB.

    Funded by: Wellcome Trust: 060551

    Journal of cellular physiology 2007;210;3;637-43

  • B-cell receptor signaling in chronic lymphocytic leukemia cells is regulated by overexpressed active protein kinase CbetaII.

    Abrams ST, Lakum T, Lin K, Jones GM, Treweeke AT, Farahani M, Hughes M, Zuzel M and Slupsky JR

    Department of Haematology, University of Liverpool, UK.

    Signals through the B-cell antigen receptor (BCR) are important for the survival of chronic lymphocytic leukemia (CLL) cells. Therefore, factors that influence these signals have important pathophysiological roles in this disease. One key mediator of BCR signaling is protein kinase C beta (PKCbeta), which regulates the activation of I-kappaB kinases and the deactivation of Bruton tyrosine kinase within the signaling pathways initiated by BCR engagement. The present study demonstrates that overexpression of the PKCbetaII isoform is a feature of CLL cells and that activity of this enzyme strongly correlates with CLL cell response to BCR engagement. Thus, intracellular Ca2+ release and increases in cell survival after BCR cross-linking were significantly greater in CLL patients with low levels than in CLL patients with high levels of active PKCbetaII. Furthermore, BCR-induced Ca2+ fluxes could be restored in CLL patients with high levels of active PKCbetaII by pretreating the cells with the PKCbeta-specific inhibitor LY379196. Conversely, BCR-mediated intracellular Ca2+ release could be inhibited in CLL cells with low levels of active PKCbetaII by pretreatment with the PKC agonist bryostatin. Taken together, these results demonstrate that overexpressed active PKCbetaII plays a role in the regulation and outcome of BCR signals that can be important for the progression of CLL.

    Blood 2007;109;3;1193-201

  • Protein kinase C-beta 1 gene variants are not associated with autism in the Irish population.

    Yang MS, Cochrane L, Conroy J, Hawi Z, Fitzgerald M, Gallagher L and Gill M

    Department of Psychiatry, Trinity Centre for Health Sciences, St James's Hospital, Dublin, Ireland, and Laboratory of Disorder Genes, Chongqing University of Medical Sciences, Chongqing, PR China. yangm@tcd.ie

    Some evidences indicate that protein kinase C-beta 1 (PRKCB1) gene may be a predisposition locus of autism. A recent study reported evidence of association between autism and two haplotypes made up of six noncoding single nucleotide polymorphisms in the PRKCB1. To attempt replication of their findings, we examined the same six single nucleotide polymorphisms of PRKCB1 in 171 Irish autism trios. The haploview program was used to calculate D' as a measure of linkage disequilibrium. The transmission disequilibrium test for single nucleotide polymorphism markers and haplotypes was carried out using the TDTPHASE and PDTPHASE from the UNPHASED version 2.404 programs. Transmission disequilibrium test analysis showed no evidence of association for any of the six single nucleotide polymorphisms at the PRKCB1 that we studied, or any of their haplotypes. Our data do not support the finding that the PRKCB1 gene variants contribute risk for the development of autism.

    Funded by: Wellcome Trust

    Psychiatric genetics 2007;17;1;39-41

  • aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb.

    Smith CA, Lau KM, Rahmani Z, Dho SE, Brothers G, She YM, Berry DM, Bonneil E, Thibault P, Schweisguth F, Le Borgne R and McGlade CJ

    Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

    In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. Here, we identify in vivo phosphorylation sites of mammalian Numb and show that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions.

    The EMBO journal 2007;26;2;468-80

  • Crystal structure of Rac1 bound to its effector phospholipase C-beta2.

    Jezyk MR, Snyder JT, Gershberg S, Worthylake DK, Harden TK and Sondek J

    Department of Biochemistry and Biophysics The University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.

    Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-beta2 (PLC-beta2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-beta2 to optimize its orientation for substrate membranes. Gbetagamma also engages the PH domain to activate PLC-beta2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-delta, the PH domain of PLC-beta2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1-PLC-beta2 complex reveals determinants that dictate selectivity of PLC-beta isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-beta2 abrogate its stimulation by Rac1 but not by Gbetagamma, allowing for functional dissection of this integral signaling node.

    Funded by: NIGMS NIH HHS: GM 057391

    Nature structural & molecular biology 2006;13;12;1135-40

  • Protein kinase C beta and delta isoenzymes mediate cholesterol accumulation in PMA-activated macrophages.

    Ma HT, Lin WW, Zhao B, Wu WT, Huang W, Li Y, Jones NL and Kruth HS

    Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1422, USA.

    Previously, we showed that PMA activation of human monocyte-derived macrophages stimulates macropinocytosis (i.e., fluid-phase endocytosis) of LDL and transforms these macrophages into foam cells. The current study aimed to learn which PKC isoenzymes mediate cholesterol accumulation in PMA-activated human macrophages incubated with LDL. Cholesterol accumulation by PMA-activated macrophages incubated with LDL was nearly completely inhibited (>85%) by the pan PKC inhibitors Go6850, Go6983, and RO 32-0432, but only was inhibited about 50% by the classical group PKC inhibitor, Go6976. This indicated that cholesterol accumulation was mediated by both a classical group and some other PKC isoenzyme. PKC beta was determined to be the classical group isoenzyme that mediated PMA-stimulated cholesterol accumulation. A pseudosubstrate myristoylated peptide inhibitor of PKC alpha and beta showed partial inhibition (congruent with 50%) of cholesterol accumulation. However, a small molecule inhibitor of PKC alpha, HBDDE, show minimal inhibition of cholesterol accumulation while a small molecule inhibitor of PKC beta, LY333513, could completely account for the inhibition of cholesterol accumulation by the classical group PKC isoenzyme. Thus, our findings show that beta and some other PKC isoenzyme, most likely delta, mediate cholesterol accumulation when macropinocytosis of LDL is stimulated in PMA-activated human monocyte-derived macrophages.

    Biochemical and biophysical research communications 2006;349;1;214-20

  • Protein kinase Cbeta isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells.

    Suzuki S, Hayashi H, Takagi K, Kondo T, Takagi K, Ueyama J and Wakusawa S

    Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan.

    The MDR3 protein is a transporter of phosphatidylcholine on the canalicular membrane of human hepatocytes. Previously we showed that the expression of MDR3 mRNA was down-regulated by phorbol 12-myristate 13-acetate (PMA) in human Chang liver cells. In the present study, to elucidate the isoform of protein kinase C (PKC), which influences the level of MDR3 protein, we investigated the effects of PKC-specific inhibitors and antisense oligonucleotides. The level of protein decreased around 50% after treatment for 3-5 days using the dosage of PMA effective against the mRNA expression. The half-life of the MDR3 protein was estimated to be about 5 days. This decrease was antagonized by GF109203X, a non-selective inhibitor of PKCs, and Gö6976, a selective inhibitor for PKCalpha/beta. These inhibitors also suppressed the reduction in MDR3 protein. To specify the isoform of PKC, the cells were treated with antisense oligonucleotide of PKCalpha or PKCbeta. The suppressive effects on MDR3 mRNA of PMA were attenuated in antisense PKCbeta-treated cells, but those in antisense PKCalpha-treated cells were not attenuated. These suggested that PKCbeta plays a regulatory role in the expression of MDR3.

    Biochimica et biophysica acta 2006;1760;10;1552-7

  • Role of angiotensin II type 1A receptor phosphorylation, phospholipase D, and extracellular calcium in isoform-specific protein kinase C membrane translocation responses.

    Policha A, Daneshtalab N, Chen L, Dale LB, Altier C, Khosravani H, Thomas WG, Zamponi GW and Ferguson SS

    Cell Biology Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, The University of Western Ontario, 100 Perth Drive, London, Ontario N6A 5K8, Canada.

    The angiotensin II type 1A receptor (AT(1A)R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT(1A)R is coupled via G(q) to the activation of phospholipase C, the hydrolysis of phosphoinositides, release of calcium from intracellular stores, and the activation of protein kinase C (PKC). We show here that PKCbetaI and PKCbetaII exhibit different membrane translocation patterns in response to AT(1A)R agonist activation. Whereas PKCbetaII translocation to the membrane is transient, PKCbetaI displays additional translocation responses: persistent membrane localization and oscillations between the membrane and cytosol following agonist removal. The initial translocation of PKCbetaI requires the release of calcium from intracellular stores and the activation of phospholipase C, but persistent membrane localization is dependent upon extracellular calcium influx. The mutation of any of the three PKC phosphorylation consensus sites (Ser-331, Ser-338, and Ser-348) localized within the AT(1A)R C-tail significantly increases the probability that persistent increases in diacylglycerol levels and PKCbetaI translocation responses will be observed. The persistent increase in AT(1A)R-mediated diacylglycerol formation is mediated by the activation of phospholipase D. Although the persistent PKCbetaI membrane translocation response is absolutely dependent upon the PKC activity-dependent recruitment of an extracellular calcium current, it does not require the activation of phospholipase D. Taken together, we show that the patterning of AT(1A)R second messenger response patterns is regulated by heterologous desensitization and PKC isoform substrate specificity.

    The Journal of biological chemistry 2006;281;36;26340-9

  • Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation.

    Siow YL, Au-Yeung KK, Woo CW and O K

    Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7.

    Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.

    The Biochemical journal 2006;398;1;73-82

  • Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation.

    Broughman JR, Sun L, Umar S, Scott J, Sellin JH and Morris AP

    Department of Integrative Biology, University of Texas Health Science Center, Houston, TX 77030, USA.

    We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.

    Funded by: NIDDK NIH HHS: DK-59550

    American journal of physiology. Gastrointestinal and liver physiology 2006;291;2;G318-30

  • Chronic PKC-beta2 activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting.

    Broughman JR, Sun L, Umar S, Sellin JH and Morris AP

    Department of Integrative Biology, University of Texas Health Science Center, Houston, TX 77030, USA.

    We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC-beta-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80-85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (ISC) was accompanied by cytoplasmic PKC-beta2 accumulation and modest activation of PKC-beta1 and PKC-epsilon. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC-beta2 levels. Thus PKC-beta2 is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.

    Funded by: NIDDK NIH HHS: DK-59550

    American journal of physiology. Gastrointestinal and liver physiology 2006;291;2;G331-44

  • Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

    Rastaldi MP, Candiano G, Musante L, Bruschi M, Armelloni S, Rimoldi L, Tardanico R, Sanna-Cherchi S, Cherchi SS, Ferrario F, Montinaro V, Haupt R, Parodi S, Carnevali ML, Allegri L, Camussi G, Gesualdo L, Scolari F and Ghiggeri GM

    Renal Immunopathology Laboratory, Fondazione D'Amico per la Ricerca sulle Malattie Renali, Nuova Nefrologia Research Association, c/o San Carlo Borromeo Hospital, Milan, Italy.

    Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

    Kidney international 2006;70;3;477-85

  • Heterogeneity of cellular proliferation within transitional cell carcinoma: correlation of protein kinase C alpha/betaI expression and activity.

    Aaltonen V, Koivunen J, Laato M and Peltonen J

    Department of Anatomy and Cell Biology, University of Oulu, University of Oulu, Finland. vesaal@utu.fi

    A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-alpha and -betaI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-alpha in 13 out of 18 samples and -betaI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-alpha and/or -betaI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-alpha was located in the cytoplasm, whereas PKC-betaI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-alpha and -betaI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2006;54;7;795-806

  • Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G.

    Kwan HY, Huang Y and Yao X

    Li Ka Shing Institute of Health Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Hong Kong, China.

    There are two known phosphorylation-mediated inactivation mechanisms for TRPC3 channels. Protein kinase G (PKG) inactivates TRPC3 by direct phosphorylation on Thr-11 and Ser-263 of the TRPC3 proteins, and protein kinase C (PKC) inactivates TRPC3 by phosphorylation on Ser-712. In the present study, we explored the relationship between these two inactivation mechanisms of TRPC3. HEK cells were first stably transfected with a PKG-expressing construct and then transiently transfected with a TRPC3-expressing construct. Addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG), elicited a TRPC3-mediated [Ca2+]i rise in these cells. This OAG-induced rise in [Ca2+]i could be inhibited by phorbol 12-myristate 13-acetate (PMA), an agonist for PKC, in a dose-dependent manner. Importantly, point mutations at two PKG phosphorylation sites (T11A-S263Q) of TRPC3 markedly reduced the PMA inhibition. Furthermore, inhibition of PKG activity by KT5823 (1 microM) or H8 (10 microM) greatly reduced the PMA inhibition of TRPC3. These data strongly suggest that the inhibitory action of PKC on TRPC3 is partly mediated through PKG in these PKG-overexpressing cells. The importance of this scheme was also tested in vascular endothelial cells, in which PKG plays a pivotal functional role. In these cells, OAG-induced [Ca2+]i rise was inhibited by PMA, which activates PKC, and by 8-BrcGMP and S-nitroso-N-acetylpenicillamine (SNAP), both of which activate PKG. Importantly, the PMA inhibition on OAG-induced [Ca2+]i rise was significantly reduced by PKG inhibitor KT5823 (1 microM) or DT-3 (500 nM), suggesting an important role of PKG in the PMA-induced inhibition of TRPC channels in native endothelial cells.

    Journal of cellular physiology 2006;207;2;315-21

  • Polymorphisms of the protein kinase C-beta gene (PRKCB1) accelerate kidney disease in type 2 diabetes without overt proteinuria.

    Araki S, Haneda M, Sugimoto T, Isono M, Isshiki K, Kashiwagi A and Koya D

    Department of Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan. araki@belle.shiga-med.ac.jp

    Objective: We investigated the contribution of PKC-beta gene (PRKCB1) polymorphisms to diabetic kidney disease in a prospective observational follow-up study.

    A total of 364 Japanese subjects with type 2 diabetes without overt proteinuria were enrolled during 1996-1998 and followed until 2004. Five single nucleotide polymorphisms (-1504C/T, -546C/G, -348A/G, -278C/T, and -238C/G) in the promoter region of PRKCB1 were genotyped. The end points were transition from stage to stage of diabetic nephropathy as a time-to-event outcome and the annual decline rate of estimated glomerular filtration rate (eGFR) as a slope-based outcome.

    Results: During the study (median 6 years), 34 of 364 subjects (9.3%) progressed. Kaplan-Meier estimation revealed that subjects with both T allele at -1054 C/T and G allele at -546 C/G polymorphisms frequently showed transition to advanced stages of diabetic nephropathy (P = 0.015). The annual change rate in eGFR in the subjects with both alleles was also significantly higher than in others (-2.96 +/- 0.62 vs. -1.63 +/- 0.15 ml/min per 1.73 m(2)/year, P = 0.02). The estimated frequency of this risk T-G haplotype was significantly higher in the progressors who showed transition to advanced nephropathy stages (12%) than in the nonprogressors (5%) (odds ratio 2.3 [95% CI 1.0-5.2]), and it was also higher in those with accelerated decline of the Delta eGFR (> or =3 ml/min per 1.73 m(2)/year) than in those without (2.1 [1.1-3.9]).

    Conclusions: Our study indicates that PRKCB1 is a predictor for worsening of kidney disease in Japanese subjects with type 2 diabetes.

    Diabetes care 2006;29;4;864-8

  • Chondrocyte cell death mediated by reactive oxygen species-dependent activation of PKC-betaI.

    DelCarlo M and Loeser RF

    Department of Medicine, Section of Rheumatology and Department of Biochemistry, Rush Medical College, Chicago, Illinois, USA.

    Signals generated by the extracellular matrix (ECM) promote cell survival. We have shown that chondrocytes detached from their native ECM and plated without serum at low density on poly-l-lysine undergo significant cell death that is associated with the production of reactive oxygen species (ROS). No cell death or ROS production was observed when cells were plated on fibronectin under the same conditions. Cell death on poly-l-lysine could be completely inhibited with the addition of either antioxidants or inhibitors of specific protein kinase C (PKC) isoforms including PKC-betaI. PKC-betaI was noted to translocate from the cytosol to the particulate membrane after plating on poly-l-lysine, and this translocation was inhibited by the addition of an antioxidant. Time-course analyses implicated endogenous ROS production as a secondary messenger leading to PKC-betaI activation and subsequent chondrocyte cell death. Cell survival on poly-l-lysine was significantly improved in the presence of oligomycin or DIDS, suggesting that ROS production occurred via complex V of the electron transport chain of the mitochondria and that ROS were released to the cytosol via voltage-dependent anion channels. Together, these results represent a novel mechanism by which ROS can initiate cell death through the activation of PKC-betaI.

    Funded by: NIA NIH HHS: AG-16697, R01 AG016697; NIAMS NIH HHS: AR-49003, R01 AR049003, R37 AR049003

    American journal of physiology. Cell physiology 2006;290;3;C802-11

  • Insulin inhibits phagocytosis in normal human neutrophils via PKCalpha/beta-dependent priming of F-actin assembly.

    Saiepour D, Sehlin J and Oldenborg PA

    Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, 901 87, Umeå, Sweden. saiepour@acc.umu.se

    Objective: This study investigated the effects of insulin on the phagocytosis of C3bi - and IgG-opsonized yeast particles in normal human neutrophils.

    Methods: Neutrophils were incubated in different insulin concentrations for 30 minutes and stimulated by C3bi - or IgG-opsonized yeast particles. Phagocytosis was quantified by both light microscopy and FACscan flow cytometry. Laser confocal microscopy was used for quantification of F-actin levels.

    Results: Elevated insulin concentrations decreased neutrophil phagocytosis of both types of targets. This defect was shown to be in part due to a delayed phagocytosis in the presence of insulin. Following a 30 minute incubation, insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The specific PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis.

    Conclusion: Hyperinsulinemia in vitro can inhibit phagocytosis of opsonized targets in normal human neutrophils. This effect of insulin is dependent on activation of PKCalpha and/or PKCbeta, and these insulin signals may interfere with the dynamic assembly/disassembly and/or distribution of F-actin, which is required for the phagocytosis process.

    Inflammation research : official journal of the European Histamine Research Society ... [et al.] 2006;55;3;85-91

  • The hepatitis C envelope 2 protein inhibits LFA-1-transduced protein kinase C signaling for T-lymphocyte migration.

    Volkov Y, Long A, Freeley M, Golden-Mason L, O'Farrelly C, Murphy A and Kelleher D

    Dublin Molecular Medicine Centre and Department of Clinical Medicine, Trinity Centre College, Ireland. yvolkov@tcd.ie

    The ability of viruses to escape the host immune response represents a globally important problem related to a wide variety of pathogens. Hepatitis C is one of the major causes of liver disease worldwide. Clearance rates of this virus are low, and this condition normally involves a chronic inflammatory process. This raises a possibility that the virus may have developed mechanisms enabling it to evade T-cell-mediated immune surveillance. The aim of this study was to investigate the effect of the hepatitis C envelope protein E2 on LFA-1-stimulated T-cell migration and macrophage inflammatory protein (MIP-1alpha, MIP-1beta) secretion.

    Methods: T cells were stimulated through the leukocyte function-associated molecule-1 (LFA-1) receptor by incubating with either intracellular adhesion molecule 1 (ICAM-1)-Fc fusion protein or anti-LFA-1 immobilized on 8-well chamber slides. Subcellular localization of protein kinase C (PKC)-beta, CD81, and LFA-1 was determined by immunofluorescence analysis. Lipid raft formation was assessed using the Cellomics Kineticscan reader. MIP-1alpha and MIP-1beta levels were detected by enzyme-linked immunosorbent assay.

    Results: We report that the hepatitis C envelope protein E2 can dramatically inhibit T-lymphocyte motility and chemokine release induced via LFA-1 integrin ligation. We have demonstrated a novel T-lymphocyte-directed viral inhibitory mechanism involving the PKC-beta enzyme as a definitive intracellular target. E2-CD81 interaction stimulates translocation of PKC-beta to lipid rafts, thereby preventing its association with the centrosome and microtubule cytoskeleton, which is crucial to the process of T-cell migration.

    Conclusions: These studies identify a mechanism whereby the hepatitis C virus can evade the host immune response by inhibition of T-cell migration.

    Gastroenterology 2006;130;2;482-92

  • Phosphorylation of the CARMA1 linker controls NF-kappaB activation.

    Sommer K, Guo B, Pomerantz JL, Bandaranayake AD, Moreno-García ME, Ovechkina YL and Rawlings DJ

    Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington 98195, USA.

    PKC isoforms and CARMA1 play crucial roles in immunoreceptor-dependent NF-kappaB activation. We tested whether PKC-dependent phosphorylation of CARMA1 directly regulates this signaling cascade. B cell antigen receptor (BCR) engagement led to the progressive recruitment of CARMA1 into lipid rafts and to the association of CARMA1 with, and phosphorylation by, PKCbeta. Furthermore, PKCbeta interacted with the serine-rich CARMA1 linker, and both PKCbeta and PKCtheta phosphorylated identical serine residues (S564, S649, and S657) within this linker. Mutation of two of these sites ablated the functional activity of CARMA1. In contrast, deletion of the linker resulted in constitutive, receptor- and PKC-independent NF-kappaB activation. Together, our data support a model whereby CARMA1 phosphorylation controls NF-kappaB activation by triggering a shift from an inactive to an active CARMA1 conformer. This PKC-dependent switch regulates accessibility of the CARD and CC domains and controls assembly and full activation of the membrane-associated IkappaB kinase (IKK) signalosome.

    Funded by: NCI NIH HHS: CA81140; NHLBI NIH HHS: HL075453; NICHD NIH HHS: HD37091

    Immunity 2005;23;6;561-74

  • Protein kinase C alpha, betaI, and betaII isozymes regulate cytokine production in mast cells through MEKK2/ERK5-dependent and -independent pathways.

    Li G, Lucas JJ and Gelfand EW

    Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.

    Regulation of MAPK pathways by PKC isoforms was examined in murine bone marrow-derived mast cells (BMMCs). The PKCalpha, betaI, and betaII isoforms showed the most robust activation after FcepsilonR1-mediated stimulation by anti-ovalbumin specific IgE and ovalbumin (IgE-ova). PKCalpha, betaI, and betaII were all involved in activation of JNK, MEKK2, and ERK5, with differential relative contributions of each isoform to specific MAPK pathway components. BMMCs from mice lacking MEKK2 showed reduced production (50-60%) of IL-6, IL-13, and TNF-alpha after stimulation, demonstrating MEKK2-dependent and -independent pathways for cytokine production. Cytokine production was stimulated by over-expression of PKC in cells from MEKK2-deficient and wild-type mice. Activation of ERK5 did not occur in BMMCs lacking MEKK2, indicating that MEKK2-independent cytokine production was also ERK5-independent. Since MAPK modules differentially regulate mast cell functions, including degranulation and cytokine production, it is suggested that specific functions could be targeted by inhibiting specific PKC isoforms.

    Funded by: NHLBI NIH HHS: HL-36577, HL-61005

    Cellular immunology 2005;238;1;10-8

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Curcumin suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting the PKC to MAPK signaling pathways in human astroglioma cells.

    Woo MS, Jung SH, Kim SY, Hyun JW, Ko KH, Kim WK and Kim HS

    Department of Neuroscience, Ewha Institute of Neuroscience, College of Medicine, Ewha Womans University, Seoul, Republic of Korea.

    The aberrant expression of matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and angiogenesis process of brain tumor. This study has investigated the effects of curcumin on MMP-9 expression in human astroglioma cell lines. Curcumin significantly inhibited the MMP-9 enzymatic activity and protein expression that was induced by PMA. The inhibitory effect of curcumin on MMP-9 expression correlates with the decreased MMP-9 mRNA level and the suppression of MMP-9 promoter activity. The curcumin-mediated inhibition of MMP-9 gene expression appears to occur via NF-kappaB and AP-1 because their DNA binding activities were suppressed by curcumin. Furthermore, curcumin strongly repressed the PMA-induced phosphorylation of ERK, JNK, and p38 MAP kinase, which were dependent on the PKC pathway. Therefore, the inhibition of MMP-9 expression by curcumin might have therapeutic potential for controlling the growth and invasiveness of brain tumor.

    Biochemical and biophysical research communications 2005;335;4;1017-25

  • Haplotypes in the gene encoding protein kinase c-beta (PRKCB1) on chromosome 16 are associated with autism.

    Philippi A, Roschmann E, Tores F, Lindenbaum P, Benajou A, Germain-Leclerc L, Marcaillou C, Fontaine K, Vanpeene M, Roy S, Maillard S, Decaulne V, Saraiva JP, Brooks P, Rousseau F and Hager J

    IntegraGen SA, Genopole, Evry, France.

    Autism is a developmental disorder characterized by impairments in social interaction and communication associated with repetitive patterns of interest or behavior. Autism is highly influenced by genetic factors. Genome-wide linkage and candidate gene association approaches have been used to try and identify autism genes. A few loci have repeatedly been reported linked to autism. Several groups reported evidence for linkage to a region on chromosome 16p. We have applied a direct physical identity-by-descent (IBD) mapping approach to perform a high-density (0.85 megabases) genome-wide linkage scan in 116 families from the AGRE collection. Our results confirm linkage to a region on chromosome 16p with autism. High-resolution single-nucleotide polymorphism (SNP) genotyping and analysis of this region show that haplotypes in the protein kinase c-beta gene are strongly associated with autism. An independent replication of the association in a second set of 167 trio families with autism confirmed our initial findings. Overall, our data provide evidence that the PRKCB1 gene on chromosome 16p may be involved in the etiology of autism.

    Molecular psychiatry 2005;10;10;950-60

  • Increases in intracellular calcium dephosphorylate histone H3 at serine 10 in human hepatoma cells: potential role of protein phosphatase 2A-protein kinase CbetaII complex.

    Huang W, Batra S, Atkins BA, Mishra V and Mehta KD

    Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210, USA.

    We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.

    Journal of cellular physiology 2005;205;1;37-46

  • Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis.

    Roggero CM, Tomes CN, De Blas GA, Castillo J, Michaut MA, Fukuda M and Mayorga LS

    Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, 5500 Mendoza, Argentina.

    We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCbetaII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain--a key region for the function of synaptotagmins--are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.

    Developmental biology 2005;285;2;422-35

  • Genes encoding catalytic subunits of protein kinase A and risk of spina bifida.

    Zhu H, Lu W, Laurent C, Shaw GM, Lammer EJ and Finnell RH

    Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, Texas.

    Background: PRKACA and PRKACB are genes encoding the cAMP-dependent protein kinase A (PKA) catalytic subunits alpha and beta, respectively. PKA is known to be involved in embryonic development, as it down-regulates the Hedgehog (Hh) signaling pathway, which is critical to normal pattern formation and morphogenesis. The PKA-deficient mouse model, which has only a single catalytic subunit, provided intriguing evidence demonstrating a relationship between decreased PKA activity and risk for posterior neural tube defects (NTDs) in the thoracic to sacral regions of gene-knockout mice. Unlike most other mutant mouse models of NTDs, the PKA-deficient mice develop spina bifida with 100% penetrance. We hypothesized that sequence variations in human genes encoding the catalytic subunits may alter the PKA activity and similarly increase the risk of spina bifida.

    Methods: We sequenced the coding regions and the exon/intron boundaries of PRKACA and PRKACB. We also examined 3 common single-nucleotide polymorphisms (SNPs) of these 2 genes by allele discrimination.

    Results: Five sequence variants in coding region and 2 intronic sequence variants proximal to exons were detected. None of the 3 SNPs examined in the association study appeared to be associated with substantially increased risk for spina bifida.

    Conclusions: Our results did not reveal a strong association between these PKA SNPs and spina bifida risk. Nonetheless, it is important to examine the possible gene-gene interactions between PRKACA and PRKACB when evaluating the risk for NTDs, as well as genes encoding regulatory subunits of PKA. In addition, interactions with other genes such as Sonic Hedgehog (SHH) should also be considered for future investigations.

    Funded by: NINDS NIH HHS: R01 NS050249, R01 NS050249-01A1

    Birth defects research. Part A, Clinical and molecular teratology 2005;73;9;591-6

  • Protein kinase C betaII plays an essential role in dendritic cell differentiation and autoregulates its own expression.

    Cejas PJ, Carlson LM, Zhang J, Padmanabhan S, Kolonias D, Lindner I, Haley S, Boise LH and Lee KP

    Department of Microbiology and Immunology and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, Florida 33156, USA.

    Dendritic cells (DC) arise from a diverse group of hematopoietic progenitors and have marked phenotypic and functional heterogeneity. The signal transduction pathways that regulate the ability of progenitors to undergo DC differentiation, as well as the specific characteristics of the resulting DC, are only beginning to be characterized. We have found previously that activation of protein kinase C (PKC) by cytokines or phorbol esters drives normal human CD34(+) hematopoietic progenitors and myeloid leukemic blasts (KG1, K562 cell lines, and primary patient blasts) to differentiate into DC. We now report that PKC activation is also required for cytokine-driven DC differentiation from monocytes. Of the cPKC isoforms, only PKC-betaII was consistently activated by DC differentiation-inducing stimuli in normal and leukemic progenitors. Transfection of PKC-betaII into the differentiation-resistant KG1a subline restored the ability to undergo DC differentiation in a signal strength-dependent fashion as follows: 1) by development of characteristic morphology; 2) the up-regulation of DC surface markers; 3) the induction of expression of the NFkappaB family member Rel B; and 4) the potent ability to stimulate allo-T cells. Most unexpectedly, the restoration of PKC-betaII signaling in KG1a was not directly due to overexpression of the transfected classical PKC (alpha, betaII, or gamma) but rather through induction of endogenous PKC-beta gene expression by the transfected classical PKC. The mechanism of this positive autoregulation involves up-regulation of PKC-beta promoter activity by constitutive PKC signaling. These findings indicate that the regulation of PKC-betaII expression and signaling play critical roles in mediating progenitor to DC differentiation.

    Funded by: NCI NIH HHS: CA85208, CA95829

    The Journal of biological chemistry 2005;280;31;28412-23

  • PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.

    Lu W and Ziff EB

    Program in Neuroscience and Physiology, New York University School of Medicine, New York, New York 10016, USA.

    PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.

    Funded by: NIMH NIH HHS: MH067229

    Neuron 2005;47;3;407-21

  • Inhibition of IFN-alpha signaling by a PKC- and protein tyrosine phosphatase SHP-2-dependent pathway.

    Du Z, Shen Y, Yang W, Mecklenbrauker I, Neel BG and Ivashkiv LB

    Graduate Program in Immunology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA.

    Cytokine signaling by the Jak-STAT pathway is subject to complex negative regulation that limits the amplitude and duration of signal transduction. Inhibition of signaling also mediates negative crosstalk, whereby factors with opposing biological activities crossinhibit each other's function. Here, we investigated a rapidly inducible mechanism that inhibited Jak-STAT activation by IFN-alpha, a cytokine that is important for antiviral responses, growth control, and modulation of immune responses. IFN-alpha-induced signaling and gene activation were inhibited by ligation of Fc receptors and Toll-like receptors 7 and 8 in a PKCbeta-dependent manner. Neither PKCbeta nor PKCdelta influenced responses of cells treated with IFN-alpha alone. Inhibition of IFN-alpha signaling correlated with suppression of IFN-alpha-dependent antiviral responses. PKC-mediated inhibition did not require de novo gene expression but involved the recruitment of PKCbeta to the IFN-alpha receptor and interaction with protein tyrosine phosphatase SHP-2, resulting in augmented phosphatase activity. PKC-mediated inhibition of IFN-alpha signaling was abolished in SHP-2-deficient cells, demonstrating a pivotal role for SHP-2 in this inhibitory pathway. Together, our data describe a rapidly inducible, direct mechanism of inhibition of Jak-STAT signaling mediated by a PKCbeta-SHP-2 signaling pathway.

    Funded by: NCI NIH HHS: CA49152; NIAID NIH HHS: AI44938, T32 AI07621; NIDDK NIH HHS: DK50693

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;29;10267-72

  • Protein kinase C phosphorylation of the metabotropic glutamate receptor mGluR5 on Serine 839 regulates Ca2+ oscillations.

    Kim CH, Braud S, Isaac JT and Roche KW

    NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.

    Funded by: Wellcome Trust

    The Journal of biological chemistry 2005;280;27;25409-15

  • Regulation by glucagon of the rat histidase gene promoter in cultured rat hepatocytes and human hepatoblastoma cells.

    Alemán G, Ortíz V, Langley E, Tovar AR and Torres N

    Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Departamento Fisiología de la Nutrición, Universidad Nacional Autonomica de México, Mexico City, México.

    Histidase (Hal), the amino acid-degrading enzyme of histidine, is regulated by the protein content of the diet and by hormones such as glucocorticoids and glucagon. However, glucagon can activate the following two possible transduction pathways: protein kinase A (PKA) and protein kinase C (PKC). The aim of this study was to isolate the 5'-flanking region of rat Hal gene to locate possible cAMP- and glucocorticoid-responsive elements and to identify whether the activation of the Hal promoter by glucagon occurs via PKA or PKC. The results showed that glucagon was able to induce Hal expression 1.5-fold in primary hepatocytes. The addition of phorbol 12-myristate,13-acetate (PMA) and forskolin to hepatocytes increased Hal mRNA concentration by 100 and 40%, respectively. To identify the Hal gene regulatory region, a 1248-bp fragment of the 5'-region was obtained. The transcription initiation site was located at 404 bp from ATG. The sequence did not show consensus TATA-like or CAAT-like boxes in the first 100 bp upstream from the transcription start site. The promoter contained six GC rich boxes, seven putative AP1 binding sites, and four glucocorticoid-responsive elements. The putative Hal promoter region was cloned into the pGL3basic vector and transfected into HepG2 cells. Luciferase expression was significantly stimulated by glucagon (0.9-fold), forskolin (0.9-fold), PMA (2.0-fold), and dexamethasone (2.9-fold). This evidence supports that the Hal gene is turned on by glucocorticoids and by glucagon either via PKC or PKA, but prefers the PKA pathway.

    American journal of physiology. Endocrinology and metabolism 2005;289;1;E172-9

  • Activated proteinkinase B in breast cancer.

    Shcherbakov AM, Gershtein ES, Anurova OA and Kushlinskii NE

    N. N. Blokhin Russian Oncological Research Center, Russian Academy of Medical Sciences, Moscow. cesaro@front.ru.

    The content of activated (phosphorylated) form of proteinkinase B in tumors and homologous tissues of 46 patients with breast cancer was measured by enzyme immunoassay. Activity of proteinkinase B was increased in tumors of 48% patients in comparison with homologous histologically unchanged tissue. Activity of proteinkinase B in hormone-dependent mammary tumors was significantly higher than in tumor tissue from patients with negative receptor status.

    Bulletin of experimental biology and medicine 2005;139;5;608-10

  • Smad6s regulates plasminogen activator inhibitor-1 through a protein kinase C-beta-dependent up-regulation of transforming growth factor-beta.

    Berg DT, Myers LJ, Richardson MA, Sandusky G and Grinnell BW

    Division of Biotechnology Discovery Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.

    Plasminogen activator inhibitor-1 (PAI-1) is a serpin class protease inhibitor that plays a central role in the regulation of vascular function and tissue remodeling by modulating thrombosis, inflammation, and the extracellular matrix. A central mediator controlling PAI-1 is transforming growth factor-beta (TGF-beta), which induces its expression and promotes fibrosis. We have found that a unique member of the Smad family of signal transduction molecules, Smad6s, modulates the expression of PAI-1. Overexpression of Smad6s in endothelial cells increases promoter activity and PAI-1 secretion, and an antisense to Smad6s suppresses the induction of PAI-1 by TGF-beta. The effect of Smad6s on the PAI-1 promoter appeared to be the result of increase binding of the forkhead winged helix factor FoxD1 to a TGF-beta-responsive element. Furthermore, the effect of Smad6s on PAI-1 up-regulation and on FoxD1 binding was found to result from up-regulation of TGF-beta and could be inhibited by the blocking TGF-beta signaling with Smad7. The ability of Smad6s to regulate the TGF-beta promoter and subsequent PAI-1 induction was suppressed by a selective protein kinase C-beta (PKC-beta) inhibitor. Consistent with the in vitro data, we found that increased Smad6s in diseased vessels correlated with increased TGF-beta and PAI-1 levels. Overall, our results demonstrate that the level of Smad6s can alter the level of TGF-beta and the subsequent induction of PAI-1 via a FoxD1 transcription site. Furthermore, our data suggest that this process, which is up-regulated in diseased vessels, can be modulated by the inhibition of PKC-beta.

    The Journal of biological chemistry 2005;280;15;14943-7

  • Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C.

    van Baal J, de Widt J, Divecha N and van Blitterswijk WJ

    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam 1066 CX, The Netherlands.

    Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.

    The Journal of biological chemistry 2005;280;11;9870-8

  • Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation.

    Manno S, Takakuwa Y and Mohandas N

    Department of Biochemistry, Tokyo Women's Medical University, 8-1 Kawada-Cho, Shinjuku-Ku, Tokyo 162-8666, Japan.

    Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha- and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.

    Funded by: NIDDK NIH HHS: DK 26263, DK 32094

    The Journal of biological chemistry 2005;280;9;7581-7

  • Modification of PI3K- and MAPK-dependent chemotaxis in aortic vascular smooth muscle cells by protein kinase CbetaII.

    Campbell M and Trimble ER

    Department of Clinical Biochemistry and Metabolic Medicine, Queen's University Belfast, Institute of Clinical Science, Royal Victoria Hospital, Grosvenor Rd, Belfast BT12 6BJ, UK. malcolm.campbell@qub.ac.uk

    Hyperglycemia increases expression of platelet-derived growth factor (PDGF)-beta receptor and potentiates chemotaxis to PDGF-BB in human aortic vascular smooth muscle cells (VSMCs) via PI3K and ERK/MAPK signaling pathways. The purpose of this study was to determine whether increased activation of protein kinase C (PKC) isoforms had a modulatory effect on the PI3K and ERK/MAPK pathways, control of cell adhesiveness, and movement. All known PKC isoforms were assessed but only PKCalpha and PKCbetaII levels were increased in 25 mmol/L glucose. However, only PKCbetaII inhibition affected (decreased) PI3K pathway and MAPK pathway activities and inhibited PDGF-beta receptor upregulation in raised glucose, and specific MAPK inhibition was required to completely block the effect of glucose. In raised glucose conditions, activity of the ERK/MAPK pathway, PI3K pathway, and PKCbetaII were all sensitive to aldose reductase inhibition. Chemotaxis to PDGF-BB (360 pmol/L), absent in 5 mmol/L glucose, was present in raised glucose and could be blocked by PKCbetaII inhibition. Formation of lamellipodia was dependent on PI3K activation and filopodia on MAPK activation; both lamellipodia and filopodia were eliminated when PKCbetaII was inhibited. FAK phosphorylation and cell adhesion were reduced by PI3K inhibition, and although MAPK inhibition prevented chemotaxis, it did not affect FAK phosphorylation or cell adhesiveness. In conclusion, chemotaxis to PDGF-BB in 25 mmol/L glucose is PKCbetaII-dependent and requires activation of both the PI3K and MAPK pathways. Changes in cell adhesion and migration speed are mediated mainly through the PI3K pathway.

    Circulation research 2005;96;2;197-206

  • Signalling crosstalk in FGF2-mediated protection of endothelial cells from HIV-gp120.

    Langford D, Hurford R, Hashimoto M, Digicaylioglu M and Masliah E

    Department of Pathology, University of California, San Diego, La Jolla, CA, USA. tdlangford@ucsd.edu

    Background: The blood brain barrier (BBB) is the first line of defence of the central nervous system (CNS) against circulating pathogens, such as HIV. The cytotoxic HIV protein, gp120, damages endothelial cells of the BBB, thereby compromising its integrity, which may lead to migration of HIV-infected cells into the brain. Fibroblast growth factor 2 (FGF2), produced primarily by astrocytes, promotes endothelial cell fitness and angiogenesis. We hypothesized that treatment of human umbilical vein endothelial cells (HUVEC) with FGF2 would protect the cells from gp120-mediated toxicity via endothelial cell survival signalling.

    Results: Exposure of HUVEC to gp120 resulted in dose- and time-dependent cell death; whereas, pre-treatment of endothelial cells with FGF2 protected cells from gp120 angiotoxicity. Treatment of HUVEC with FGF2 resulted in dose- and time-dependent activation of the extracellular regulated kinase (ERK), with moderate effects on phosphoinositol 3 kinase (PI3K) and protein kinase B (PKB), also known as AKT, but no effects on glycogen synthase kinase 3 (GSK3beta) activity. Using pharmacological approaches, gene transfer and kinase activity assays, we show that FGF2-mediated angioprotection against gp120 toxicity is regulated by crosstalk among the ERK, PI3K-AKT and PKC signalling pathways.

    Conclusions: Taken together, these results suggest that FGF2 may play a significant role in maintaining the integrity of the BBB during the progress of HIV associated cerebral endothelial cell damage.

    Funded by: NIDA NIH HHS: DA12065, P01 DA012065; NIMH NIH HHS: K01 MH071206, MH071206, MH58164, MH59745, MH62962, P50 MH045294, R01 MH062962, R24 MH059745

    BMC neuroscience 2005;6;8

  • Sodium butyrate-mediated differentiation of colorectal cancer cells: regulation of PKCbetaII by PI 3-kinase.

    Turecková J, Vojtechová M, Kucerová D, Velek J and Tuhácková Z

    Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 16637 Prague 6, Czech Republic. turejo@img.cas.cz

    The present study focuses on a putative regulation of PKCbetaII by phosphatidylinositol-3 kinase (PI 3-kinase) in colorectal carcinoma cells; little is known about the role and activity of PKCbetaII in these cells. We examined the activity of PI 3-kinase in two adenocarcinoma cell lines, HT29 cells that differentiate only after stimulation with appropriate agents, and Caco-2 cells that can differentiate spontaneously. The activity of PI 3-kinase as well as the activity of PKCbetaII appeared to decrease only in HT29 cells in which differentiation was induced by sodium butyrate. In HT29 cells infected with recombinant adenovirus encoding constitutively active PI 3-kinase, the activity of alkaline phosphatase was almost completely blocked, and this PI 3-kinase significantly potentiated the activity of PKCbetaII in HT29 cells despite the presence of NaBT in the culture medium. On the contrary, in differentiating Caco-2 cells, the activity of PI 3-kinase was not butyrate-sensitive. In agreement with these findings, the alkaline phosphatase activity was not affected by constitutively active PI 3-kinase overexpressed in Caco-2 cells. These observations suggest that PKCbetaII is regulated by PI 3-kinase in HT29 cells and that the mechanisms of spontaneous differentiation versus butyrate-induced differentiation of adenocarcinoma cells may be different.

    International journal of molecular medicine 2005;15;2;329-35

  • Selective inhibition of juxtanuclear translocation of protein kinase C betaII by a negative feedback mechanism involving ceramide formed from the salvage pathway.

    Becker KP, Kitatani K, Idkowiak-Baldys J, Bielawski J and Hannun YA

    Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    In a previous study, we showed that protein kinase C betaII (PKC betaII) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC betaII with 4beta-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC betaII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC betaII in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC betaII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC betaII in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC betaII.

    Funded by: NHLBI NIH HHS: HL43707; NIGMS NIH HHS: GM08716

    The Journal of biological chemistry 2005;280;4;2606-12

  • Protein kinase C isoforms differentially phosphorylate human choline acetyltransferase regulating its catalytic activity.

    Dobransky T, Doherty-Kirby A, Kim AR, Brewer D, Lajoie G and Rylett RJ

    Department of Physiology, University of Western Ontario and Cell Biology Research Group, and Robarts Research Institute, London, Ontario N6A 5C1, Canada.

    Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.

    The Journal of biological chemistry 2004;279;50;52059-68

  • IL-10 production induced by HIV-1 Tat stimulation of human monocytes is dependent on the activation of PKC beta(II) and delta isozymes.

    Contreras X, Bennasser Y and Bahraoui E

    Laboratoire d'immuno-virologie des lentivirus des primates, EA3038, Université Paul Sabatier, 118, route de Narbonne, 31062 Toulouse, France.

    The effect of HIV-1 Tat protein on the production of IL-10, an immunosuppressive cytokine, was examined in human primary monocytes obtained from healthy HIV-1-negative blood donors. As expected and in agreement with our previous data, a dose-dependent induction of IL-10 was observed. In addition, we showed that this induction is mediated by the PKC pathway: in the presence of Ro 31-8220, an inhibitor of all PKC isozymes, or after 48 h of PMA treatment, Tat protein becomes unable to stimulate IL-10 production. Among the 11 PKC isozymes, eight (PKC alpha, beta(I), beta(II), delta, epsilon, eta, zeta, mu) are expressed in monocytes. In this study, by analyzing the translocation to the membrane after Tat stimulation, we showed that PKC alpha, beta(I), beta(II), delta and epsilon isozymes are activated by Tat. Moreover, by combining different approaches including selective PKC inhibitors (Gö6983, Gö6976, hispidin and rottlerin), we showed that PKC beta(II) and delta isozymes are essential for the activation of IL-10 production in human monocytes following stimulation by HIV-1 Tat protein.

    Microbes and infection 2004;6;13;1182-90

  • Protein kinase CbetaII regulates its own expression in rat intestinal epithelial cells and the colonic epithelium in vivo.

    Liu Y, Su W, Thompson EA, Leitges M, Murray NR and Fields AP

    Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224, USA.

    Protein kinase C betaII (PKCbetaII) is induced early during colon carcinogenesis. Transgenic mice expressing elevated PKCbetaII in the colonic epithelium (transgenic PKCbetaII mice) exhibit hyperproliferation and enhanced colon carcinogenesis. Here we demonstrate that nullizygous PKCbeta (PKCbetaKO) mice are highly resistant to azoxymethane (AOM)-induced preneoplastic lesions, aberrant crypt foci. However, reexpression of PKCbetaII in the colon of PKCbetaKO mice by transgenesis restores susceptibility to AOM-induced colon carcinogenesis. Expression of human PKCbetaII in rat intestinal epithelial (RIE) cells induces expression of endogenous rat PKCbetaII mRNA and protein. Induction of PKCbetaII is dependent upon catalytically active PKCbetaII and does not appear to involve changes in alternative splicing of the PKCbeta gene. Two human PKCbeta promoter constructs are activated by expression of PKCbetaII in RIE cells. Both PKCbeta promoter activity and PKCbetaII mRNA levels are inhibited by the MEK1 and -2 inhibitor U0126, but not the Cox-2 inhibitor celecoxib in RIE/PKCbetaII cells. PKCbeta promoter activity correlates directly with expression of endogenous PKCbetaII mRNA and protein in HT29 and HCT116 human colon cancer cell lines. PKCbeta promoter activity and PKCbetaII mRNA expression in HCT116 cells are inhibited by the selective PKCbeta inhibitor LY317615 and by U0126, demonstrating autoregulation of PKCbetaII expression. Transgenic PKCbetaII mice exhibit specific induction of endogenous PKCbetaII, but not its splice variant PKCbetaI, in the colonic epithelium in vivo. Taken together, our results demonstrate that 1) expression of PKCbetaII in the colonic epithelium is both necessary and sufficient to confer susceptibility to AOM-induced colon carcinogenesis in transgenic mice, 2) PKCbetaII regulates its own expression in RIE and human colon cancer cells in vitro and in the colonic epithelium in vivo, and 3) PKCbetaII autoregulation is mediated through a MEK-dependent signaling pathway in RIE/PKCbetaII and HCT116 colon cancer cells.

    Funded by: NCI NIH HHS: CA81436

    The Journal of biological chemistry 2004;279;44;45556-63

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Polymorphisms in the 5'-upstream region of the PKCbeta gene in Japanese patients with Type 2 diabetes.

    Ikeda Y, Suehiro T, Osaki F, Tsuzura S, Kumon Y and Hashimoto K

    Department of Endocrinology, Metabolism and Nephrology, Kochi Medical School, Japan. ikeday@kochi-ms.ac.jp

    Aims: Protein kinase C (PKC), a serine/threonine kinase, is known to be activated in various tissues under hyperglycaemic conditions. Notably, PKCbeta, a member of the conventional PKC group, is the predominant isoform detected in vascular tissues and could be involved in the development of diabetic vascular complications. In the present study, we investigated genetic variations in the 5'-upstream region of the PKCbeta gene to assess their possible relation to vascular complications in diabetic patients.

    Methods: Variations upstream from the PKCbeta gene (-1066/+256) were examined in 60 Type 2 diabetic patients using a cycle sequencing method. Screening of detected variations was performed in 204 Type 2 diabetic patients and 160 healthy controls.

    Results: Five single nucleotide polymorphisms; C(-238)G, C(-287)T, A(-348)G, C(-546)G, and C(-853)T, were identified in the upstream region. The C(-287)T and A(-348)G polymorphisms were in perfect linkage disequilibrium. There were no significant differences in genotype or allele frequencies of the five polymorphisms among the diabetic patients and healthy subjects. However, both -238GG and -287CC (-348GG) homozygotes showed significantly higher frequencies of macrovascular disease compared with patients with other genotypes. Further, an electrophoretic mobility shift assay revealed that the -238G fragment had a five-fold higher affinity for transcription factor Sp1 when compared with -238C.

    Conclusions: The C(-238)G and C(-287)T-A(-348)G polymorphisms in the 5'-upstream region of the PKCbeta gene may have an effect on the susceptibility of diabetic vascular complications through an alteration of tissue PKCbeta density or function.

    Diabetic medicine : a journal of the British Diabetic Association 2004;21;10;1113-20

  • High expression of PKC-MAPK pathway mRNAs correlates with glomerular lesions in human diabetic nephropathy.

    Toyoda M, Suzuki D, Honma M, Uehara G, Sakai T, Umezono T and Sakai H

    Division of Nephrology and Metabolism, Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.

    Background: Activation of protein kinase C (PKC) is a major signaling pathway for transforming growth factor (TGF)-beta to induce extracellular matrix (ECM) production in diabetic nephropathy (DN). PKC also activates mitogen-activated protein kinase (MAPK), which is called the PKC-MAPK pathway. The PKC-MAPK pathway is probably responsible for PKC-related abnormalities in diabetic glomeruli. To confirm the involvement of this pathway, we determined the localization and expression of mRNAs in glomeruli by in situ hybridization method.

    Methods: In the present study, we examined expression of PKCbeta1, MAPK/ERK kinase (MEK) 1, MEK2, extracellular signal-regulated protein kinase (ERK) 1, ERK2, and TGF-beta1 mRNAs using renal tissue samples from kidneys affected by DN (N= 21) and from normal human kidney (NHK; N= 6). We also performed an immunohistochemical study using anti-phosphorylated MEK1/2 (P-MEK) and ERK1/2 (P-ERK) antibodies. The glomerular severity of DN was classified into three groups according to mesangial expansion: D1 (N= 4), D2 (N= 13), and D3 (N= 4). We analyzed differences and correlations between variables.

    Results: In the glomeruli, the number of cells that stained for these mRNAs in DN was significantly higher than in NHK. The expression of PKC-MAPK pathway mRNAs tended to be inversely proportional to the degree of mesangial expansion. The P-MEK and P-ERK signal intensity were parallel to its mRNA expression pattern. Furthermore, there were significant correlations among the P-MEK, P-ERK signal intensity, PKCbeta1 mRNA expression.

    Conclusion: Our results suggest that high expression of PKC-MAPK pathway mRNAs plays an important role in the development and/or progression of early tissue damage in DN.

    Kidney international 2004;66;3;1107-14

  • The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-beta on melanosomes.

    Park HY, Wu H, Killoran CE and Gilchrest BA

    Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118, USA. hypark@bu.edu

    Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, alpha, beta, delta, epsilon and zeta isoforms of PKC are expressed, but uniquely PKC-beta activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-beta phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-beta, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-beta, revealed abundant PKC-beta, whereas PKC-alpha was not detected when immunoblot analysis was performed using antibody against PKC-alpha. Activation of PKC in melanocytes increased the level of PKC-beta co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-beta in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-beta and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-beta on the melanosome membrane, allowing PKC-beta to phosphorylate tyrosinase.

    Funded by: NCI NIH HHS: CA R01 72763

    Journal of cell science 2004;117;Pt 16;3659-68

  • Isoenzyme-specific translocation of protein kinase C (PKC)betaII and not PKCbetaI to a juxtanuclear subset of recycling endosomes: involvement of phospholipase D.

    Becker KP and Hannun YA

    Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.

    Elucidation of isoenzyme-specific functions of individual protein kinase C (PKC) isoenzymes has emerged as an important goal in the study of this family of kinases, but this task has been complicated by modest substrate specificity and high homology among the individual members of each PKC subfamily. The classical PKCbetaI and PKCbetaII isoenzymes provide a unique opportunity because they are the alternatively spliced products of the beta gene and are 100% identical except for the last 50 of 52 amino acids. In this study, it is shown that green fluorescent protein-tagged PKCbetaII and not PKCbetaI translocates to a recently described juxtanuclear site of localization for PKCalpha and PKCbetaII isoenzymes that arises with sustained stimulation of PKC. Mechanistically, translocation of PKCbetaII to the juxtanuclear region required kinase activity. PKCbetaII, but not PKCbetaI, was found to activate phospholipase D within this time frame. Inhibitors of phospholipase D (1-butanol and a dominant negative construct) prevented the translocation of PKCbetaII to the juxtanuclear region but not to the plasma membrane, thus demonstrating a role for phospholipase D in the juxtanuclear translocation of PKCbetaII. Taken together, these results define specific biochemical and cellular actions of PKCbetaII when compared with PKCbetaI.

    Funded by: NHLBI NIH HHS: HL 43707

    The Journal of biological chemistry 2004;279;27;28251-6

  • PKCdelta associates with and is involved in the phosphorylation of RasGRP3 in response to phorbol esters.

    Brodie C, Steinhart R, Kazimirsky G, Rubinfeld H, Hyman T, Ayres JN, Hur GM, Toth A, Yang D, Garfield SH, Stone JC and Blumberg PM

    Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel. chaya@mail.biu.ac.il

    RasGRP is a family of guanine nucleotide exchange factors that activate small GTPases and contain a C1 domain similar to the one present in protein kinase C (PKC). In this study, we examined the interaction of RasGRP3 and PKC in response to the phorbol ester PMA. In Chinese hamster ovary or LN-229 cells heterologously expressing RasGRP3, phorbol 12-myristate 13-acetate (PMA) induced translocation of RasGRP3 to the perinuclear region and a decrease in the electrophoretic mobility of RasGRP3. The mobility shift was associated with phosphorylation of RasGRP3 on serine residues and seemed to be PKCdelta-dependent because it was blocked by the PKCdelta inhibitor rottlerin as well as by a PKCdelta kinase-dead mutant. Using coimmunoprecipitation, we found that PMA induced the physical association of RasGRP3 with PKCdelta and, using in situ methods, we showed colocalization of PKCdelta and RasGRP3 in the perinuclear region. PKCdelta phosphorylated RasGRP3 in vitro. Previous studies suggest that ectopic expression of RasGRP3 increases activation of Erk1/2. We found that overexpression of either PKCdelta or RasGRP3 increased the activation of Erk1/2 by PMA. In contrast, coexpression of PKCdelta and RasGRP3 yielded a level of phosphorylation of Erk1/2 similar to that of control vector cells. Our results suggest that PKCdelta may act as an upstream kinase associating with and phosphorylating RasGRP3 in response to PMA. The interaction between RasGRP3 and PKCdelta points to the existence of complex cross-talk between various members of the phorbol ester receptors which can have important impact on major signal transduction pathways and cellular processes induced by phorbol esters or DAG

    Molecular pharmacology 2004;66;1;76-84

  • Protein kinase C (PKC) betaII induces cell invasion through a Ras/Mek-, PKC iota/Rac 1-dependent signaling pathway.

    Zhang J, Anastasiadis PZ, Liu Y, Thompson EA and Fields AP

    Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224, USA.

    Protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. Expression of PKCbetaII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCbetaII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCbetaII. Here we show that RIE/PKCbetaII cells acquire an invasive phenotype that is blocked by the PKCbeta inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCbetaII, indicating that PKCbetaII activity is required for the invasive phenotype. PKCbetaII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCbetaII cells. PKCbetaII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKCiota. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCbetaII-mediated invasion. Taken together, our results define a novel PKCbetaII --> Ras --> PKCiota /Rac1 --> Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCbetaII promotes colon carcinogenesis.

    The Journal of biological chemistry 2004;279;21;22118-23

  • Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry.

    Hassel S, Eichner A, Yakymovych M, Hellman U, Knaus P and Souchelnytskyi S

    Ludwig Institute for Cancer Research, Uppsala, Sweden.

    Bone morphogenetic proteins (BMP) are polypeptide growth factors that regulate cell differentiation and proliferation. BMPs bind to type I and type II serine/threonine kinase receptors to initiate intracellular signalling. BMPR-II is the type II receptor, its mutations lead to hereditary pulmonary hypertension, and knockout of Bmpr-II results in early embryonic lethality. To identify novel interacting proteins and explore signalling pathways that can be initiated by BMPR-II, we performed glutathione-S-transferase (GST) pull-down assays with BMPR-II protein constructs fused to GST and extracts of mouse myoblast C2C12 cells. We generated three constructs which contain different parts of the cytoplasmic region of BMPR-II: full-length cytoplasmic part of BMPR-II, only the kinase domain, or only the C-terminal tail of BMPR-II. Proteins which formed complexes with these BMPR-II constructs were analyzed by two-dimensional gel electrophoresis (2-D GE), and specifically interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). We identified 33 interacting proteins; 11 proteins interacted with the C-terminal tail of BMPR-II, 4 with full-length BMPR-II, and 18 with a short form of the receptor with a deleted tail. Fourteen proteins have assigned functions in various signalling processes, suggesting links of BMP signalling to regulation of MAP kinase pathway, apoptosis, transcription, PKCss, and PKA. Five of the identified proteins are components of the cytoskeleton, and four are enzymes involved in metabolism, e.g., processing of estrogens or lipids. We confirmed interaction of PKC beta and CtBP with BMPR-II using immunodetection. We showed that the C-terminal tail of BMPR-II provides binding sites for a number of regulatory proteins that may initiate Smad-independent signalling.

    Proteomics 2004;4;5;1346-58

  • Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton.

    Harrison RE, Sikorski BA and Jongstra J

    Cell Biology Programme, The Hospital for Sick Children Research Institute, Toronto, Ontario, M5G 1X8, Canada.

    The identification and characterization of scaffold and targeting proteins of the ERK/MAP kinase pathway is important to understand the function and intracellular organization of this pathway. The F-actin binding protein leukocyte-specific protein 1 (LSP1) binds to PKCbetaI and expression of B-LSP1, an LSP1 truncate containing the PKCbetaI binding residues, inhibits anti-IgM-induced translocation of PKCbetaI to the plasma membrane and anti-IgM-induced activation of ERK2. To understand the role of LSP1 in the regulation of PKCbetaI-dependent ERK2 activation, we investigated whether LSP1 interacts with ERK/MAP kinase pathway components and targets these proteins to the actin cytoskeleton. We show that LSP1 associates with the ERK scaffold protein KSR and with MEK1 and ERK2. LSP1-associated MEK1 is activated by anti-IgM treatment and this activation is inhibited by expression of B-LSP1, suggesting that the activation of LSP1-associated MEK1 is PKCbetaI dependent. Confocal microscopy showed that LSP1 targets KSR, MEK1 and ERK2 to peripheral actin filaments. Thus our data show that LSP1 is a cytoskeletal targeting protein for the ERK/MAP kinase pathway and support a model in which MEK1 and ERK2 are organized in a cytoskeletal signaling complex together with KSR, PKCbetaI and LSP1 and are activated by anti-IgM in a PKCbetaI-dependent manner.

    Journal of cell science 2004;117;Pt 10;2151-7

  • Ki-1/57 interacts with RACK1 and is a substrate for the phosphorylation by phorbol 12-myristate 13-acetate-activated protein kinase C.

    Nery FC, Passos DO, Garcia VS and Kobarg J

    Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10.000, C.P. 6192, 13084-971 Campinas, SP, Brasil.

    Ki-1/57, the 57-kDa human protein antigen recognized by the CD30 antibody Ki-1, is a cytoplasmic and nuclear protein that is phosphorylated on serine and threonine residues. When isolated from the Hodgkin's lymphoma analogous cell line L540 Ki-1/57 co-immunoprecipitated with a Thr/Ser protein kinase activity. It has been also found to interact with hyaluronic acid and has therefore been termed intracellular IHABP4 (hyaluronan-binding protein 4). Recent studies demonstrated, however, that Ki-1/57 engages in specific interaction with the chromo-helicase-DNA-binding domain protein 3, a nuclear protein involved in chromatin remodeling and transcription regulation. We used the yeast two-hybrid system to find proteins interacting with Ki-1/57 and identified the adaptor protein RACK1 (receptor of activated kinase 1). Next, we confirmed this interaction in vitro and in vivo, performed detailed mapping studies of the interaction sites of Ki-1/57 and RACK-1, and demonstrated that Ki-1/57 also co-precipitates with protein kinase C (PKC) when isolated from phorbol 12-myristate 13-acetate (PMA)-activated L540 tumor cells and is a substrate for PKC phosphorylation in vitro and in vivo. Interestingly, the interaction of Ki-1/57 with RACK1 is abolished upon activation of L540 cells with PMA, which results in the phosphorylation of Ki-1/57 and its exit from the nucleus. Taken together, our data suggest that Ki-1/57 forms a stable complex with RACK-1 in unstimulated cells and upon PMA stimulation gets phosphorylated on threonine residues located at its extreme C terminus. These events associate Ki-1/57 with the RACK1/PKC pathway and may be important for the regulation of its cellular functions.

    The Journal of biological chemistry 2004;279;12;11444-55

  • PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers.

    Banan A, Zhang LJ, Farhadi A, Fields JZ, Shaikh M and Keshavarzian A

    Division of Digestive Diseases, Department of Internal Medicine, Department of Pharmacology, and Department of Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612, USA. ali_banan@rush.edu

    Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.

    Funded by: NIAAA NIH HHS: AA-13745; NIDDK NIH HHS: DK-60511

    American journal of physiology. Cell physiology 2004;286;3;C723-38

  • Centrosomal anchoring of protein kinase C betaII by pericentrin controls microtubule organization, spindle function, and cytokinesis.

    Chen D, Purohit A, Halilovic E, Doxsey SJ and Newton AC

    Department of Pharmacology, School of Medicine, University of California at San Diego, La Jolla, California 92093-0640, USA.

    Location is a critical determinant in dictating the cellular function of protein kinase C (PKC). Scaffold proteins contribute to localization by poising PKC at specific intracellular sites. Using a yeast two-hybrid screen, we identified the centrosomal protein pericentrin as a scaffold that tethers PKC betaII to centrosomes. Co-immunoprecipitation studies reveal that the native proteins interact in cells. Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494-593. Immunofluorescence analysis reveals that endogenous PKC betaII colocalizes with pericentrin at centrosomes. Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure. Overexpression of this disrupting fragment has no effect in cells lacking PKC betaII, indicating a specific regulatory role of this isozyme in centrosome function. These results reveal a novel role for PKC betaII in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes.

    Funded by: NIDDK NIH HHS: DK54441; NIGMS NIH HHS: GM51994

    The Journal of biological chemistry 2004;279;6;4829-39

  • Effects of the antiandrogen flutamide on the expression of protein kinase C isoenzymes in LNCaP and PC3 human prostate cancer cells.

    Montalvo L, Carmena MJ, Bolaños O, Rodríguez-Henche N, Sánchez-Chapado M and Prieto JC

    Departamento de Bioquímica y Biologia Molecular, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain.

    Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (alpha, beta1, epsilon, zeta) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 microM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of alpha, beta1 and zeta, but not epsilon, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCbeta1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.

    Bioscience reports 2004;24;1;11-21

  • A chromatin immunoprecipitation screen reveals protein kinase Cbeta as a direct RUNX1 target gene.

    Hug BA, Ahmed N, Robbins JA and Lazar MA

    Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA.

    RUNX1 (also known as AML1) is a DNA-binding transcription factor that functions as a tumor suppressor and developmental determinant in hematopoietic cells. Target promoters have been identified primarily through the use of differential expression strategies and candidate gene approaches but not biochemical screens. Using a chromatin immunoprecipitation screen, we identified protein kinase Cbeta as a direct RUNX1 target gene and demonstrate that endogenous RUNX1 binds the chromatinized protein kinase Cbeta promoter of U937 cells. A phylogenetically conserved RUNX1-binding site within the PKCbeta promoter binds RUNX1 in electrophoretic mobility shift analyses and confers RUNX1 responsiveness on a heterologous promoter. Changes in RUNX1 activity affect endogenous protein kinase Cbeta expression, and a dominant-negative form of RUNX1 protects U937 cells from apoptotic stimuli previously shown to be dependent on protein kinase Cbeta. This protection can be reversed by the ectopic expression of protein kinase Cbeta. Together these findings demonstrate that protein kinase Cbeta is a direct, downstream target of RUNX1 and links RUNX1 to a myeloid apoptotic pathway.

    The Journal of biological chemistry 2004;279;2;825-30

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • cPKC-dependent sequestration of membrane-recycling components in a subset of recycling endosomes.

    Becker KP and Hannun YA

    Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    In addition to the classical role of protein kinase C (PKC) as a mediator of transmembrane signals initiated at the plasma membrane, there is also significant evidence to suggest that a more sustained PKC activity is necessary for a variety of long term cellular responses. To date, the subcellular localization of PKC during sustained activation has not been extensively studied. We report here that long term activation of PKC (1 h) leads to the selective translocation of classical PKC isoenzymes, alpha and betaII, to a juxtanuclear compartment. Juxtanuclear translocation of PKC required an intact C1 and C2 domain, and occurred in a microtubule-dependent manner. This juxtanuclear compartment was localized close to the Golgi complex but displayed no overlap with Golgi markers, and was resistant to dispersal with Golgi disrupting agents, brefeldin A and nocodazole. Further characterization revealed that PKCalpha and betaII translocated to a compartment that colocalized with the small GTPase, rab11, which is a marker for the subset of recycling endosomes concentrated around the microtubule-organizing center/centrosome. Analysis of the functional consequence of cPKC translocation on membrane recycling demonstrated a cPKC-dependent sequestration of transferrin, a marker of membrane recycling, in the cPKC compartment. These results identify a novel site for cPKC translocation and define a novel function for the sustained activation of PKCalpha and betaII in regulation of recycling components.

    Funded by: NHLBI NIH HHS: HL 43707

    The Journal of biological chemistry 2003;278;52;52747-54

  • Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2.

    Lorenz K, Lohse MJ and Quitterer U

    Institut für Pharmakologie und Toxikologie, Versbacher Strasse 9, D-97078 Würzburg, Germany.

    Feedback inhibition is a fundamental principle in signal transduction allowing rapid adaptation to different stimuli. In mammalian cells, the major feedback inhibitor for G-protein-coupled receptors (GPCR) is G-protein-coupled receptor kinase 2 (GRK-2), which phosphorylates activated receptors, uncouples them from G proteins and initiates their internalization. The functions of GRK-2 are indispensable and need to be tightly controlled. Dysregulation promotes disorders such as hypertension or heart failure. In our search for a control mechanism for this vital kinase, here we show that the Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of GRK-2. After stimulation of GPCR, RKIP dissociates from its known target, Raf-1 (refs 6-8), to associate with GRK-2 and block its activity. This switch is triggered by protein kinase C (PKC)-dependent phosphorylation of the RKIP on serine 153. The data delineate a new principle in signal transduction: by activating PKC, the incoming receptor signal is enhanced both by removing an inhibitor from Raf-1 and by blocking receptor internalization. A physiological role for this mechanism is shown in cardiomyocytes in which the downregulation of RKIP restrains beta-adrenergic signalling and contractile activity.

    Nature 2003;426;6966;574-9

  • Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly.

    Ceci M, Gaviraghi C, Gorrini C, Sala LA, Offenhäuser N, Marchisio PC and Biffo S

    Molecular Histology Unit, DIBIT-HSR, 20132 Milano, Italy.

    The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.

    Nature 2003;426;6966;579-84

  • Protein kinase C-beta-mediated complex formation between tyrosinase and TRP-1.

    Wu H and Park HY

    Department of Hematology/Oncology, Massachusetts General Hospital, Boston, MA 02114, USA.

    Tyrosinase, the key enzyme in melanogenesis, is activated when protein kinase C-beta (PKC-beta) phosphorylates the serine residues at amino acid positions 505 and 509. To further elucidate the mechanism by which phosphorylation of tyrosinase by PKC-beta leads to the activation of tyrosinase, a possible complex formation between phosphorylated tyrosinase and tyrosinase related protein-1 (TRP-1), a melanogenic protein suggested to influence tyrosinase activity, was investigated. Non-denaturing gel electrophoresis of melanocyte lysate revealed two molecular weight forms of TRP-1 and a monoclonal antibody against TRP-1 co-immunoprecipitated tyrosinase and TRP-1, suggesting that TRP-1 may be complexed with tyrosinase. Activation of PKC by treating melanocytes with phorbol 12,13-dibutyrate (PDBu) increased the level of tyrosinase co-immunoprecipitated with TRP-1; whereas a selective PKC inhibitor bisindolylmaleimide inhibited PDBu-induced increase in the level of tyrosinase co-immunoprecipitated with TRP-1. These results suggest that phosphorylation of tyrosinase by PKC-beta induces a complex formation between tyrosinase and TRP-1.

    Funded by: NCI NIH HHS: CA R0172763

    Biochemical and biophysical research communications 2003;311;4;948-53

  • Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.

    Chen W, ten Berge D, Brown J, Ahn S, Hu LA, Miller WE, Caron MG, Barak LS, Nusse R and Lefkowitz RJ

    Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

    Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.

    Funded by: NHLBI NIH HHS: HL 16037, HL 61365; NINDS NIH HHS: NS 19576

    Science (New York, N.Y.) 2003;301;5638;1391-4

  • Physiological regulation of Munc18/nSec1 phosphorylation on serine-313.

    Craig TJ, Evans GJ and Morgan A

    Department of Physiology, University of Liverpool, Liverpool, UK.

    Increased protein phosphorylation enhances exocytosis in most secretory cell types, including neurones. However, the molecular mechanisms by which this occurs and the specific protein targets remain unclear. Munc18-1/nSec1 is essential for exocytosis in neurones, and is known to be phosphorylated by protein kinase C (PKC) in vitro at Ser-313. This phosphorylation has been shown to decrease its affinity for syntaxin, and to alter the kinetics of exocytosis in chromaffin cells. However, there are no data on the physiological regulation of Ser-313 phosphorylation. Using phospho-Ser-313-specific antisera, we demonstrate here that Ser-313 is phosphorylated in intact and permeabilized chromaffin cells in response to histamine and Ca2+ respectively. Furthermore, Ser-313 is rapidly and transiently phosphorylated in intact synaptosomes in response to depolarization by KCl treatment or by 4-aminopyridine, and by the metabotropic glutamate receptor agonist dihydroxyphenylglycine. PKC was identified as the kinase, and PP1 and PP2B as the phosphatases responsible for regulating Ser-313 phosphorylation. As phosphorylation of nSec1 on Ser-313 affects the rate of transmitter release in chromaffin cells, the demonstration here that this phosphorylation event occurs in neurones suggests that synaptic neurotransmitter release may be similarly regulated by nSec1 phosphorylation. Furthermore, such changes in release kinetics are associated with long-term potentiation and depression, thus implicating nSec1 phosphorylation as a potential regulatory mechanism underlying presynaptic plasticity.

    Journal of neurochemistry 2003;86;6;1450-7

  • Protein kinase Cbeta1, a major regulator of TCR-CD28-activated signal transduction leading to IL-2 gene transcription and secretion.

    Dreikhausen UE, Gorf K, Resch K and Szamel M

    Institute of Pharmacology, Medical School Hannover, 30623 Hannover, Germany.

    The aim of this study was to investigate the influence of protein kinase C (PKC) alpha and beta on the TCR-CD28-stimulated protein kinase cascades participating in regulation of IL-2 gene transcription and secretion. Inhibition of the synthesis of PKCalpha and beta by specific phosphorothioate-modified antisense oligonucleotides (ODN) resulted in suppression of phosphorylation and activation of Raf-1, mitogen-activated extracellular-regulated kinase kinases and extracellular-regulated kinases in stimulated Jurkat T cells. Furthermore, a marked reduction of IkappaB kinase-alpha-catalyzed IkappaBalpha phosphorylation was observed in both PKCalpha- and beta-specific antisense oligonucleotide-treated cells. In sharp contrast, TCR-CD28-stimulated phosphorylation and activation of the Jun-N-terminal kinase (JNK) cascade was specifically suppressed upon treatment with PKCbeta-specific antisense ODN, suggesting that PKCbeta was a specific upstream regulator of the JNK protein kinase cascade. Significant inhibition of high-affinity NF-AT binding and transactivation, IL-2 gene expression, reduction of IL-2 mRNA synthesis, and, most impressively, a complete suppression of IL-2 secretion were observed in PKCbeta antisense ODN-treated cells. The data indicate a highly specific function of PKCbeta for regulation of TCR-CD28 induced-signaling, IL-2 gene expression and secretion in Jurkat T cells.

    International immunology 2003;15;9;1089-98

  • Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3.

    Teixeira C, Stang SL, Zheng Y, Beswick NS and Stone JC

    Department of Biochemistry, University of Alberta, Edmonton, Canada.

    Members of the RasGRP family of Ras activators have C1 domains that bind diacylglycerol (DAG) and DAG analogs such as the tumor-promoting phorbol esters. RasGRP members could be responsible for some of the DAG signaling processes that have previously been attributed to protein kinase C (PKC). We found that RasGRP3 is selectively expressed in B cells, suggesting that RasGRP3 might function downstream of the B-cell receptor (BCR). Indeed, stimulation of Ramos B cells with the DAG analog phorbol ester myristate (PMA) results in the association of RasGRP3 with the membrane fraction. However, we also made the unexpected observation that RasGRP3 is phosphorylated, coincident with Ras activation after stimulation. When inhibitors of PKC are present, Ras activation is attenuated, and this attenuation correlates with an inhibition of RasGRP3 phosphorylation. RasGRP3 is phosphorylated in vitro by PKC-theta and PKC-beta2. When ectopically coexpressed in HEK-293 cells, a dominant-activated mutant of PKC-theta phosphorylates RasGRP3 and enhances Ras-Erk signaling. These results provide the first indication for a functional interaction between a RasGRP family member and a dissimilar DAG binding protein. A convergent DAG signaling system could be important in fine-tuning Ras signaling during B-cell development or during the humoral immune response.

    Blood 2003;102;4;1414-20

  • Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betaII mediated by phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent pathways.

    Helliwell PA, Rumsby MG and Kellett GL

    Department of Biology (Area 3), University of York, York YO10 5YW, United Kingdom.

    Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) betaII activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC betaII phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brush-border membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC betaII recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC betaII by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250+ kDa. PMA increased the level of the PKC betaII 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC betaII turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC betaII and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.

    The Journal of biological chemistry 2003;278;31;28644-50

  • Identification of a common risk haplotype for diabetic nephropathy at the protein kinase C-beta1 (PRKCB1) gene locus.

    Araki S, Ng DP, Krolewski B, Wyrwicz L, Rogus JJ, Canani L, Makita Y, Haneda M, Warram JH and Krolewski AS

    Section on Genetics and Epidemiology, Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA.

    Abnormal activation of protein kinase C-beta isoforms in the diabetic state has been implicated in the development of diabetic nephropathy. It is thus plausible that DNA sequence differences in the protein kinase C-beta1 gene (PRKCB1), which encodes both betaI and betaII isoforms, may influence susceptibility to nephropathy. Nine single-nucleotide polymorphisms (SNP) in PRKCB1 were tested for association with diabetic nephropathy in type I diabetes mellitus, by using both case-control and family-study designs. Allele and genotype distributions of two SNP in the promoter (--1504C/T and --546C/G) differed significantly between case patients and control patients (P < 0.05). These associations were particularly strong with diabetes mellitus duration of <24 yr (P = 0.002). The risk of diabetic nephropathy was higher among carriers of the T allele of the --1504C/T SNP, compared with noncarriers (odds ratio, 2.54; 95% confidence interval, 1.39 to 4.62), and among carriers of the G allele of the --546C/G SNP (odds ratio, 2.45; 95% confidence interval, 1.37 to 4.38). Among individuals with diabetes mellitus duration of >/==" BORDER="0">24 yr, these two SNP were not associated with diabetic nephropathy. These positive findings were confirmed by using the family-based transmission disequilibrium test. The T-G haplotype, with both risk alleles, was transmitted more frequently than expected from heterozygous parents to offspring who developed diabetic nephropathy during the first 24 yr of diabetes mellitus. It is concluded that DNA sequence differences in the promoter of PRKCB1 contribute to diabetic nephropathy susceptibility in type I diabetes mellitus.

    Funded by: NIDDK NIH HHS: DK41526, DK53534

    Journal of the American Society of Nephrology : JASN 2003;14;8;2015-24

  • Protein kinase C beta is required for human monocyte chemotaxis to MCP-1.

    Carnevale KA and Cathcart MK

    Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.

    Funded by: NHLBI NIH HHS: HL61971

    The Journal of biological chemistry 2003;278;28;25317-22

  • Protein kinase C beta2-dependent phosphorylation of core 2 GlcNAc-T promotes leukocyte-endothelial cell adhesion: a mechanism underlying capillary occlusion in diabetic retinopathy.

    Chibber R, Ben-Mahmud BM, Mann GE, Zhang JJ and Kohner EM

    Centre for Cardiovascular Biology & Medicine, Guy's, King's and St. Thomas' School of Biomedical Sciences, King's College London, 2nd Floor New Hunt's House, London SE1 1UL, U.K. rakesh.chibber@kcl.ac.uk

    Increased leukocyte-endothelial cell adhesion is a key early event in the development of retinopathy and atherogenesis in diabetic patients. We recently reported that raised activity of glycosylating enzyme [beta]1,6 acetylglucosaminyltransferase (core 2 GlcNAc-T) is responsible for increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. Here, we demonstrate that elevated glucose increases the activity of core 2 GlcNAc-T and adhesion of human leukocytes to retinal capillary endothelial cells, in a dose-dependent manner, through diabetes-activated serine/threonine protein kinase C beta2 (PKCbeta2)-dependent phosphorylation. This regulatory mechanism, involving phosphorylation of core 2 GlcNAc-T, is also present in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients. Inhibition of PKCbeta2 activation with the specific inhibitor, LY379196, attenuated serine phosphorylation of core 2 GlcNAc-T and prevented increased leukocyte-endothelial cell adhesion. Raised activity of core 2 GlcNAc-T was associated with a threefold increase in O-linked glycosylation of P-selectin glycoprotein ligand-1 on the surface of leukocytes of diabetic patients compared with age-matched control subjects. PKCbeta2-dependent phosphorylation of core 2 GlcNAc-T may thus represent a novel regulatory mechanism for activation of this key enzyme in mediating increased leukocyte-endothelial cell adhesion and capillary occlusion in diabetic retinopathy.

    Diabetes 2003;52;6;1519-27

  • Neural cell adhesion molecule (NCAM) association with PKCbeta2 via betaI spectrin is implicated in NCAM-mediated neurite outgrowth.

    Leshchyns'ka I, Sytnyk V, Morrow JS and Schachner M

    Zentrum für Molekulare Neurobiologie, Universität Hamburg, Germany.

    In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100-insoluble complexes with betaI spectrin. Heteromeric spectrin (alphaIbetaI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the betaI spectrin fragment encompassing second and third spectrin repeats (betaI2-3). In NCAM120-transfected cells, betaI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-beta-cyclodextrin disrupts the NCAM120-spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180-betaI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCbeta2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180-spectrin-PKCbeta2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative betaI2-3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCbeta2 and NCAM140 or NCAM180. Redistribution of PKCbeta2 to NCAM-spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with betaI2-3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM-spectrin-PKCbeta2 complex is necessary for NCAM-mediated neurite outgrowth.

    The Journal of cell biology 2003;161;3;625-39

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Threonine phosphorylation sites in the beta 2 and beta 7 leukocyte integrin polypeptides.

    Hilden TJ, Valmu L, Kärkkäinen S and Gahmberg CG

    Department of Biosciences, Division of Biochemistry, and Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

    The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The beta(2) leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758-760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the beta(2) integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, beta(7), has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused beta(7) integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the beta(7) subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in beta(2) phosphorylation.

    Journal of immunology (Baltimore, Md. : 1950) 2003;170;8;4170-7

  • Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein.

    Corbit KC, Trakul N, Eves EM, Diaz B, Marshall M and Rosner MR

    Department of Neurobiology, Pharmacology and Physiology, and Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637, USA.

    Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.

    Funded by: NCI NIH HHS: 5T32CA09594; NIGMS NIH HHS: T32GM07151; NINDS NIH HHS: NS38846

    The Journal of biological chemistry 2003;278;15;13061-8

  • Phosphorylation of serine 1106 in the catalytic domain of topoisomerase II alpha regulates enzymatic activity and drug sensitivity.

    Chikamori K, Grabowski DR, Kinter M, Willard BB, Yadav S, Aebersold RH, Bukowski RM, Hickson ID, Andersen AH, Ganapathi R and Ganapathi MK

    Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.

    Funded by: NCI NIH HHS: R01 CA74939; NIDDK NIH HHS: R01 DK56917

    The Journal of biological chemistry 2003;278;15;12696-702

  • Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase.

    Yang XL, Zhang YL, Lai ZS, Xing FY and Liu YH

    Institute of Gastroenterology, NanFang Hospital, First Military Medical University 510515, Guangzhou, Guangdong Province, China. foru3000@hotmail.com

    Aim: To study the detail mechanism of interaction between PKC and GRK(2) and the effect of GRK(2) on activity of PKC.

    Methods: The cDNA of pleckstrin homology (PH) domain located in GRK(2) residue 548 to 660 was amplified by PCR with the mRNA of human GRK(2) (beta1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK(2) PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK(2) PH domain fusion protein, BTK (Bruton's tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK(2) in culture mammalian cells (6 cell lines: PC-3, MDCK, SGC7901, Jurkat cell etc.) was determined by SDS-PAGE and Co-immunoprecipitation. The binding of GRK(2) PH domain, GST-GRK(2) PH domain fusion protein and BTK PH domain to PKC in Vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK(2) to PKC was also detected by western blot and Co-immunoprecipitation.

    Results: The binding of GRK(2) PH domain to PKC in Vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK(2) PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK(2) PH domain.

    Conclusion: GRK(2) can bind with PKC and down-regulated PKC activity.

    World journal of gastroenterology 2003;9;4;800-3

  • Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis.

    Barclay JW, Craig TJ, Fisher RJ, Ciufo LF, Evans GJ, Morgan A and Burgoyne RD

    Physiological Laboratory, University of Liverpool, Crown Street, United Kingdom.

    Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.

    The Journal of biological chemistry 2003;278;12;10538-45

  • Functional association of cytokine-induced SH2 protein and protein kinase C in activated T cells.

    Chen S, Anderson PO, Li L, Sjögren HO, Wang P and Li SL

    Immunology Group, Institute of Cell and Molecular Sciences, St Barts & The Royal London School of Medicine, Queen Mary University of London, London EC1A 7BE, UK.

    TCR signaling is mediated by intracellular signaling molecules and nuclear transcription factors, which are tightly regulated by interaction with regulatory proteins such as Grb2 and SLAP. We reported recently that TCR stimulation induces the expression of cytokine-induced SH2 protein (CIS). The expression of CIS promotes TCR-mediated activation. We have now found specific interactions between CIS and activated protein kinase C (PKC) alpha, beta and theta in TCR-stimulated T cells. CIS was shown by in vitro kinase assay to associate with activated PKC. In CIS-expressing T cells isolated from CIS-transgenic mice, the amount of activated PKC associated with CIS was found to increase following TCR stimulation. By immunohistochemical analysis, CIS was also found to co-localize with PKCtheta at the plasma membrane of activated T cells. In addition to the interaction and intracellular co-localization of the CIS and PKC, an increase in the activation of AP-1 and NF-kappaB was noted in CIS-expressing T cells, after stimulation by either anti-CD3/CD28 or phorbol myristate acetate + ionomycin. These results suggest that CIS regulates PKC activation, and that this may be important for the activation of both the AP-1 and NF-kappaB pathways in TCR signaling.

    International immunology 2003;15;3;403-9

  • Interaction of "readthrough" acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behavior.

    Birikh KR, Sklan EH, Shoham S and Soreq H

    Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Israel.

    Behavioral reactions to stress are altered in numerous psychiatric and neurodegenerative syndromes, but the corresponding molecular processes and signal transduction pathways are yet unknown. Here, we report that, in mice, the stress-induced splice variant of acetylcholinesterase, AChE-R, interacts intraneuronally with the scaffold protein RACK1 and through it, with its target, protein kinase CbetaII (PKCbetaII), which is known to be involved in fear conditioning. In stress-responsive brain regions of normal FVBN mice, the mild stress of i.p. injection increased AChE and PKCbetaII levels in a manner suppressible by antisense prevention of AChE-R accumulation. Injection stress also prolonged conflict between escape and hiding in the emergence into an open field test. Moreover, transgenic FVBN mice overexpressing AChE-R displayed prolonged delay to emerge into another field (fear-induced behavioral inhibition), associated with chronically intensified neuronal colabeling of RACK1 and PKCbetaII in stress-responsive brain regions. These findings are consistent with the hypothesis that stress-associated changes in cholinergic gene expression regulate neuronal PKCbetaII functioning, promoting fear-induced conflict behavior after stress.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;1;283-8

  • PH domain of G protein-coupled receptor kinase-2 binds to protein kinase C (PKC) and negatively regulates activity of PKC kinase.

    Ji S, Liu X, Li S, Shen L, Li F, Wang J, Han J and Yao L

    Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi'an, Shaaxi, 710032. PR China.

    G protein-coupled receptor kinase-2 (GRK),also known as beta1-adrenergic receptor kinase(beta-ARK1), plays an important role in agonist-induced desensitization of the beta-adrenergic receptors. Activation of protein kinase C (PKC) is able to stimulate phosphorylation and activation of GRKs and induce desensitization of G protein-coupled receptor. However, detail mechanism of interaction between PKC and GRK2 and the effect of GRK2 on activity of PKC remain unknown. Pleckstrin homology (PH) domain is a kind of functionally domain containing about 120 amino acids, which exists on many protein molecules that involve in cellular signal transduction. A PH domain located in GRK2 residue 548 to 660 may play a significant role in mediating interaction between PKC and GRK2. In present study, we revealed that PKC could associate with PH domain of GRK2 in pull-down assay in vitro. Co-immunoprecipitation displayed binding of PKC to GRK2 in intact Jurkat cells after prolonged stimulation of epinephrine. Assay of PKC beta1 kinase activity indicated that the binding of the PH domain of GRK2 to PKC beta 1 could down-regulate activity of PKC beta 1 kinase. Thus, GRK2 may play a negative feedback regulatory role on PKCbeta1 activity in interaction between GRK2 and PKCbeta 1.

    Frontiers in bioscience : a journal and virtual library 2003;8;a34-9

  • Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C.

    Young SH, Wu SV and Rozengurt E

    Department of Medicine, Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, David Geffen School of Medicine, UCLA-CURE Digestive Diseases Research Center and Molecular Biology Institute, UCLA, 90095-1786, USA.

    We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.

    Funded by: NCI NIH HHS: P50 CA90388; NIDDK NIH HHS: DK 17294, DK 55003, DK 56930

    The Journal of biological chemistry 2002;277;49;46871-6

  • Signaling pathways triggered by HIV-1 Tat in human monocytes to induce TNF-alpha.

    Bennasser Y, Badou A, Tkaczuk J and Bahraoui E

    Laboratoire d'Immuno-Virologie, EA 3038, Université Paul Sabatier 118, route de Narbonne, 31062, Toulouse Cedex, France.

    In this study we investigated the signaling pathways triggered by Tat in human monocyte to induce TNF-alpha. In monocytes, the calcium, the PKA, and the PKC pathways are highly implicated in the expression of cytokine genes. Thus, these three major signaling pathways were investigated. Our data show that (i) PKC and calcium pathways are required for TNF-alpha production, whereas the PKA pathway seems to be not involved; (ii) downstream from PKC, activation of NFkappaB is essential while ERK1/2 MAP kinases, even though activated by Tat, are not directly involved in the pathway signaling leading to TNF-alpha production.

    Virology 2002;303;1;174-80

  • Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells.

    Pinton P, Tsuboi T, Ainscow EK, Pozzan T, Rizzuto R and Rutter GA

    Henry Wellcome Signalling Laboratories and the Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom.

    The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.

    The Journal of biological chemistry 2002;277;40;37702-10

  • Biphasic regulation of plasminogen activator/inhibitor by LDL in mesangial cells.

    Song CY, Kim BC, Hong HK, Kim BK, Kim YS and Lee HS

    Department of Pathology, Seoul National University College of Medicine, Korea.

    Lipid abnormalities and dysregulation of the plasminogen activator (PA)/plasmin system may be involved in the development of glomerulosclerosis. We investigated the effects of low-density lipoprotein (LDL) on PA inhibitor-1 (PAI-1), urokinase-type PA (uPA), and tissue-type PA (tPA) in relationship to protein kinase C (PKC) in cultured human mesangial cells (HMC). LDL (200 microg/ml) induced two peaks of PKC activation at hours 0.25 and 6, with translocation of PKC-alpha, -beta(1), and -delta from cytosol to the membrane. The second increase in PKC activity gradually decreased to the control value by hour 18. LDL downregulated 2.4-kb PAI-1, uPA, and tPA mRNA expression within 6 h of incubation with HMC. On the other hand, after 12-48 h, LDL-treated cells showed a significant increase in PAI-1, tPA, and uPA mRNA levels. LDL induced up to a twofold increase in PAI-1 antigen levels in the extracellular matrix of HMC after 24-48 h as well as increased PA inhibitory activity in the culture medium. Analysis of the adhesion plaques from cells incubated with LDL for 48 h by zymography showed increased intensity of lysis near molecular weights of approximately 55,000 and 100,000. LDL slightly increased tPA release at hours 24 and 48 but did not increase PA activity in culture medium. The stimulatory effects of LDL on PAI-1, tPA, and uPA gene regulation in HMC were blocked by the inhibition of PKC using GF-109203X 12 h after treatment with LDL or downregulation of PKC using phorbol myristate acetate. In summary, LDL regulates PAI-1, uPA, and tPA in biphasic patterns in HMC, and the upregulation of PAI-1, uPA, and tPA after long-term LDL exposure seems to be mediated by a delayed PKC activation associated with an increased PA inhibitory activity. These results suggest that LDL, after prolonged incubations with HMC, causes a PA/inhibitor imbalance favoring accumulation of matrix.

    American journal of physiology. Renal physiology 2002;283;3;F423-30

  • The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C.

    Gao T and Newton AC

    Department of Pharmacology, University of California, La Jolla, California 92093-0640, USA.

    Heat shock proteins play central roles in ensuring the correct folding and maturation of cellular proteins. Here we show that the heat shock protein Hsp70 has a novel role in prolonging the lifetime of activated protein kinase C. We identified Hsp70 in a screen for binding partners for the carboxyl terminus of protein kinase C. Co-immunoprecipitation experiments revealed that Hsp70 specifically binds the unphosphorylated turn motif (Thr(641) in protein kinase C beta II), one of three priming sites phosphorylated during the maturation of protein kinase C family members. The interaction of Hsp70 with protein kinase C can be abolished in vivo by co-expression of fusion proteins encoding the carboxyl terminus of protein kinase C or the carboxyl terminus of Hsp70. Pulse-chase experiments reveal that Hsp70 does not regulate the maturation of protein kinase C: the rate of processing by phosphorylation is the same in the presence or absence of disrupting constructs. Rather, Hsp70 prolongs the lifetime of mature protein kinase C; disruption of the interaction promotes the accumulation of matured and then dephosphorylated protein kinase C in the detergent-insoluble fraction of cells. Furthermore, studies with K562 cells reveal that disruption of the interaction with Hsp70 slows the protein kinase C beta II-mediated recovery of cells from PMA-induced growth arrest. Last, we show that other members of the AGC superfamily (Akt/protein kinase B and protein kinase A) also bind Hsp70 via their unphosphorylated turn motifs. Our data are consistent with a model in which Hsp70 binds the dephosphorylated carboxyl terminus of mature protein kinase C, thus stabilizing the protein and allowing re-phosphorylation of the enzyme. Disruption of this interaction prevents re-phosphorylation and targets the enzyme for down-regulation.

    Funded by: NIDDK NIH HHS: P01 DK54441; NIGMS NIH HHS: R01 GM43154

    The Journal of biological chemistry 2002;277;35;31585-92

  • PKC-beta controls I kappa B kinase lipid raft recruitment and activation in response to BCR signaling.

    Su TT, Guo B, Kawakami Y, Sommer K, Chae K, Humphries LA, Kato RM, Kang S, Patrone L, Wall R, Teitell M, Leitges M, Kawakami T and Rawlings DJ

    The Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.

    NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies.

    Funded by: NCI NIH HHS: CA09120, CA74929, CA81140; NIAID NIH HHS: AI33617, AI38348; NICHD NIH HHS: HD37091; NIGMS NIH HHS: GM08042

    Nature immunology 2002;3;8;780-6

  • Sequence determinants on the NR2A and NR2B subunits of NMDA receptor responsible for specificity of phosphorylation by CaMKII.

    Mayadevi M, Praseeda M, Kumar KS and Omkumar RV

    Rajiv Gandhi Centre for Biotechnology, Jagathy, Thiruvananthapuam, Kerala-695014, India.

    Calcium/calmodulin-dependent protein kinase type II (CaMKII) and NMDA-type glutamate receptor (NMDAR) are neuronal proteins involved in learning and memory. CaMKII binds to the NR2B subunit of NMDAR in more than one mode, a stable association involving a noncatalytic site on CaMKII and an enzyme-substrate mode of interaction by its catalytic site. The latter binding results in phosphorylation of serine-1303 on NR2B. We have investigated this binding by studying the kinetics of phosphorylation of synthetic peptides harboring nested sequences of the phosphorylation site motif. We find that residues 1292-1297 of NR2B enhance the affinity of the catalytic site-mediated binding of CaMKII to the minimal phosphorylation site motif, 1298-1308 of NR2B, as evident from measurements of K(m) values for phosphorylation. However, CaMKII shows decreased affinity towards the closely related NR2A subunit due to an -Ile-Asn- motif present as a natural insertion in the analogous sequence on NR2A.

    Biochimica et biophysica acta 2002;1598;1-2;40-5

  • PC phosphorylation increases the ability of AFAP-110 to cross-link actin filaments.

    Qian Y, Baisden JM, Cherezova L, Summy JM, Guappone-Koay A, Shi X, Mast T, Pustula J, Zot HG, Mazloum N, Lee MY and Flynn DC

    The Mary Babb Randolph Cancer Center and the Department of Microbiology and Immunology, West Virginia University, Morgantown, West Virginia 26506-9300, USA.

    The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src(527F) can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.

    Funded by: NCI NIH HHS: CA60731, R01 CA060731

    Molecular biology of the cell 2002;13;7;2311-22

  • Phosphorylation of p47phox sites by PKC alpha, beta II, delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation.

    Fontayne A, Dang PM, Gougerot-Pocidalo MA and El-Benna J

    Institut National de la Santé et de la Recherche Médicale U-479, Centre Hospitalier Universitaire Xavier Bichat, Paris, France.

    Production of superoxide anions by the multicomponent enzyme of human neutrophil NADPH oxidase is accompanied by extensive phosphorylation of p47(phox), one of its cytosolic components. p47(phox) is an excellent substrate for protein kinase C (PKC), but the respective contribution of each PKC isoform to this process is not clearly defined. In this study, we found that PKC isoforms known to be present in human neutrophils (PKC alpha, beta, delta, and zeta) phosphorylate p47(phox) in a time- and concentration-dependent manner, with apparent K(m) values of 10.33, 3.37, 2.37, and 2.13 microM for PKC alpha, beta II, delta, and zeta, respectively. Phosphopeptide mapping of p47(phox) showed that, as opposed to PKC zeta, PKC alpha, beta II, and delta are able to phosphorylate all the major PKC sites. The use of p47(phox) mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC alpha, beta II, and delta. Comparison of the intensity of phosphopeptides suggests that Ser 328 is the most phosphorylated serine. The ability of each PKC isoform to induce p47(phox) to associate with p22(phox) was tested by using an overlay technique; the results showed that all the PKC isoforms that were studied induce p47(phox) binding to the cytosolic fragment of p22(phox). In addition, PKC alpha, beta II, delta, and zeta were able to induce production of superoxide anions in a cell-free system using recombinant cytosolic proteins. Surprisingly, PKC zeta, which phosphorylates a subset of selective p47(phox) sites, induced stronger activation of the NADPH oxidase. Taken together, these results suggest that PKC alpha, beta II, delta, and zeta expressed in human neutrophils can individually phosphorylate p47(phox) and induce both its translocation and NADPH oxidase activation. In addition, phosphorylation of some serines could have an inhibitory effect on oxidase activation.

    Biochemistry 2002;41;24;7743-50

  • Protein kinase C beta controls nuclear factor kappaB activation in B cells through selective regulation of the IkappaB kinase alpha.

    Saijo K, Mecklenbräuker I, Santana A, Leitger M, Schmedt C and Tarakhovsky A

    Laboratory of Lymphocyte Signaling, Rockefeller University, New York, NY 10021, USA. saijok@mail.rockefeller.edu

    Activation of the nuclear factor (NF)-kappaB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-kappaB is critically dependent on serine phosphorylation of the IkappaB protein by the multi-component IkappaB kinase (IKK) containing two catalytic subunits (IKKalpha and IKKbeta) and one regulatory subunit (IKKgamma). Using mice deficient for protein kinase C beta (PKCbeta) we show an essential role of PKCbeta in the phosphorylation of IKKalpha and the subsequent activation of NF-kappaB in B cells. Defective IKKalpha phosphorylation correlates with impaired B cell antigen receptor-mediated induction of the pro-survival protein Bcl-xL. Lack of IKKalpha phosphorylation and defective NF-kappaB induction in the absence of PKCbeta explains the similarity in immunodeficiencies caused by PKCbeta or IKKalpha ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Bruton's tyrosine kinase (Btk), which regulates the activity of NF-kappaB and PKCbeta, suggests PKCbeta as a likely serine/threonine kinase component of the Btk-dependent NF-kappaB activating signal transduction chain downstream of the BCR.

    The Journal of experimental medicine 2002;195;12;1647-52

  • The beta 1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells.

    Banan A, Fields JZ, Farhadi A, Talmage DA, Zhang L and Keshavarzian A

    Department of Internal Medicine, Section of Gastroenterology and Nutrition, Rush University Medical Center, 1725 West Harrison, Chicago, IL 60612, USA. ali_banan@rush.edu

    Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca(2+) normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-beta1 (PKC-beta1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-beta1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H(2)O(2) +/- EGF, PKC, or Ca(2+) modulators. Other cells were transfected to over-express PKC-beta1 or to inhibit its expression and then pretreated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H(2)O(2). In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-beta1, enhanced (45)Ca(2+) efflux, normalized Ca(2+), decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably over-expressing PKC-beta1 (+3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-beta1. Antisense inhibition of PKC-beta1 expression (-90%) prevented all measures of EGF protection. Inhibitors of Ca(2+)-ATPase prevented EGF protection in N cells as well as protective synergism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-beta1. EGF normalizes Ca(2+) by enhancing Ca(2+) efflux through PKC-beta1. We have identified novel biologic functions, protection of actin and Ca(2+) homeostasis, among the classical isoforms of PKC.

    The Journal of pharmacology and experimental therapeutics 2002;301;3;852-66

  • The use of fluorescent phorbol esters in studies of protein kinase C-membrane interactions.

    Slater SJ, Ho C and Stubbs CD

    Department of Anatomy, Pathology and Cell Biology, Thomas Jefferson University, Room 271 JAH, 1020 Locust St., Philadelphia, PA 19107, USA.

    The family of protein kinase C (PKC) isozymes belongs to a growing class of proteins that become active by associating with membranes containing anionic phospholipids, such as phosphatidylserine. Depending on the particular PKC isoform, this process is mediated by Ca(2+)-binding to a C2 domain and interaction of activators such as 1,2-diacyl-sn-glycerol or phorbol esters with tandem C1 domains. This cooperation between the C1 and C2 domains in inducing the association of PKC with lipid membranes provides the energy for a conformational change that consists of the release of a pseudosubstrate sequence from the active site, culminating in activation. Thus, the properties of the interactions of the C1 and C2 domains with membranes, both as isolated domains, and as modules in the full length PKC isoforms, have been the subject of intense scrutiny. Here, we review the findings of studies in which fluorescent phorbol esters have been utilized to probe the properties of the C1 domains of PKC with respect to the interaction with activators, the subsequent interaction with membranes, and the role of the activating conformational change that leads to activation.

    Funded by: NIAAA NIH HHS: AA07186, AA07215, AA07465, AA08022

    Chemistry and physics of lipids 2002;116;1-2;75-91

  • Protein kinase C-beta II Is an apoptotic lamin kinase in polyomavirus-transformed, etoposide-treated pyF111 rat fibroblasts.

    Chiarini A, Whitfield JF, Armato U and Dal Pra I

    Histology and Embryology Unit, Department of Biomedical and Surgical Sciences, Medical School, University of Verona, Verona I-37134, Italy.

    The role of protein kinase C-beta(II) (PKC-beta(II)) in etoposide (VP-16)-induced apoptosis was studied using polyomavirus-transformed pyF111 rat fibroblasts in which PKC-beta(II) specific activity in the nuclear membrane (NM) doubled and the enzyme was cleaved into catalytic fragments. No PKC-beta(II) complexes with lamin B1 and/or active caspases were immunoprecipitable from the NM of proliferating untreated cells, but large complexes of PKC-beta(II) holoprotein and its catalytic fragments with lamin B1, active caspase-3 and -6, and inactive phospho-CDK-1, but not PKC-beta(I) or PKC-delta, could be immunoprecipitated from the NM of VP-16-treated cells, suggesting that PKC-beta(II) is an apoptotic lamin kinase. By 30 min after normal nuclei were mixed with cytoplasms from VP-16-treated, but not untreated, cells, PKC-beta(II) holoprotein had moved from the apoptotic cytoplasm to the normal NM, and lamin B1 was phosphorylated before cleavage by caspase-6. Lamin B1 phosphorylation was partly reduced, but its cleavage was completely prevented, despite the presence of active caspase-6, by adding a selective PKC-betas inhibitor, hispidin, to the apoptotic cytoplasms. Thus, a PKC-beta(II) response to VP-16 seems necessary for lamin B1 cleavage by caspase-6 and nuclear lamina dissolution in apoptosing pyF111 fibroblasts. The possibility of PKC-beta(II) being an apoptotic lamin kinase in these cells was further suggested by lamin B1-bound PKC-delta being inactive or only slightly active and by PKC-alpha not combining with the lamin.

    The Journal of biological chemistry 2002;277;21;18827-39

  • Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction.

    Dai F, Yu L, He H, Chen Y, Yu J, Yang Y, Xu Y, Ling W and Zhao S

    State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, 220 Handan Road, Shanghai 200433, People's Republic of China.

    Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.

    Biochemical and biophysical research communications 2002;293;4;1191-6

  • Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry.

    Ruse CI, Willard B, Jin JP, Haas T, Kinter M and Bond M

    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

    We have developed and validated a method that uses liquid chromatography/electrospray ionization-mass spectrometry to quantify site-specific protein phosphorylation. The method uses selected ion monitoring to determine the chromatographic peak areas of specific tryptic peptides from the protein of interest. The extent of phosphorylation is determined from the ratio of the phosphopeptide peak area to the peak area of an unmodified reference peptide that acts as internal standard, correcting for variations in protein amounts and peptide recovery in the digest preparation procedure. As a result, we refer to this protocol as the native reference peptide method. Mole of phosphate at the selected site per mole of protein is obtained from this ratio, using calibration curves of synthetic peptides to determine relative responses. Our method begins with protein separation by SDS-PAGE and is carried out on amounts of peptide produced by an in-gel digestion of single Coomassie blue-stained bands. To illustrate the utility of the method and provide validation, we used cardiac troponin I as analyte and monitored the time course of a protein kinase C betaII reaction. Those analyses appropriately demonstrate the time-dependent increase of phosphorylation at a PKC-preferred site, Ser44 in the peptide 41ISASPR45 and the concomitant consumption of the nonphosphorylated peptide. We believe that this method provides a novel tool to directly measure specific phosphorylation sites in proteins in different physiological states and expect that the method will be adaptable not only to a variety of samples types (i.e., culture cells, tissues, etc.) but to a variety of posttranslation modifications as well.

    Funded by: NHLBI NIH HHS: R01 HL56256, T32 HL07653

    Analytical chemistry 2002;74;7;1658-64

  • Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway.

    Fang X, Yu S, Tanyi JL, Lu Y, Woodgett JR and Mills GB

    Department of Molecular Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

    Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor beta subunit (PDGFRbeta) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRbeta mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cgamma (PLCgamma) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRbeta. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCgamma-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.

    Funded by: NCI NIH HHS: CA64602, CA82716, P01 CA064602, R01 CA082716

    Molecular and cellular biology 2002;22;7;2099-110

  • HIV-1 Tat protein induces interleukin-10 in human peripheral blood monocytes: involvement of protein kinase C-betaII and -delta.

    Bennasser Y and Bahraoui E

    Laboratoire d'Immuno-Virologie, EA 3038, Université Paul Sabatier, 31062 Toulouse, France.

    In HIV-infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with the disease progression toward AIDS. We have previously shown that HIV-1 Tat induces IL-10 production by human monocytes via a protein kinase C (PKC) -dependent pathway. Here we show that PKC activation by Tat is essential for IL-10 induction. Among the eight PKC isoforms present in human monocytes, we investigated which isoform(s) plays this crucial role in Tat-mediated IL-10 production and show that 1) Tat can activate PKC-alpha, PKC-betaII, PKC-delta, and PKC-epsilon, 2) of these four potential candidates, only PKC-betaII, PKC-delta, and PKC-epsilon are activated by the active domain Tat 1-45, which is responsible for IL-10 production and depleted by long-term exposure to PMA, which abolishes Tat-mediated IL-10 production, 3) whereas selective inhibition of PKC-alpha and PKC-epsilon by specific antisense oligonucleotides has no effect on Tat-mediated IL-10 induction, inhibition of either PKC-betaII or PKC-delta partially inhibits IL-10 production; and 4) the simultaneous inhibition of PKC-betaII and PKC-delta totally inhibits Tat-mediated IL-10. Altogether, these results suggest that the induction of IL-10 by Tat is strictly dependent on the PKC-delta and -betaII isoforms.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2002;16;6;546-54

  • Nanomolar amyloid beta protein activates a specific PKC isoform mediating phosphorylation of MARCKS in Neuro2A cells.

    Tanimukai S, Hasegawa H, Nakai M, Yagi K, Hirai M, Saito N, Taniguchi T, Terashima A, Yasuda M, Kawamata T and Tanaka C

    Hyogo Institute for Aging Brain and Cognitive Disorders, 520 Saisho-ko, Himeji 670-0981, Japan.

    Myristoylated alanine-rich C kinase substrate (MARCKS), a protein associated with cell growth, neurosecretion and macrophage activation, is activated by protein kinase C (PKC) phosphorylation. We reported that amyloid beta protein (Abeta) activated MARCKS through a tyrosine kinase and PKC-delta in rat cultured microglia. Here we report that Abeta signaling pathway through a specific PKC isoform is involved in the phosphorylation of MARCKS in Neuro2A cells. Selective PKC inhibitors but not tyrosine kinase inhibitors significantly inhibited the phosphorylation of MARCKS induced by Abeta. Abeta selectively activated PKC-alpha among the four PKC isoforms localized in Neuro2A cells. PKC-alpha activated by Abeta directly phosphorylated a recombinant MARCKS in vitro, Translocation of PKC-alpha from the cytoplasm to the membrane and accumulation of phospho-MARCKS in the cytoplasm were induced by Abeta. These results suggest involvement of a phosphoinositide signaling system through PKC-alpha in the phosphorylation of MARCKS in neurons, an event which may be associated with mechanisms underlying neurotrophic and neurotoxic effects of Abeta.

    Neuroreport 2002;13;4;549-53

  • Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

    Dellis O, Gangloff SC, Paulais M, Tondelier D, Rona JP, Brouillard F, Bouteau F, Guenounou M and Teulon J

    Electrophysiologie des Membranes, Université Denis Diderot (Paris7), 2 place Jussieu, 75251 Paris cedex 05, France. odellis@moka.ccr.jussieu.fr

    The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by Gö 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.

    The Journal of biological chemistry 2002;277;8;6044-50

  • Characterization of protein kinase C beta isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization.

    Suzuma K, Takahara N, Suzuma I, Isshiki K, Ueki K, Leitges M, Aiello LP and King GL

    Research Division, Joslin Diabetes Center, Boston, MA 02215, USA.

    Retinal neovascularization is a major cause of blindness and requires the activities of several signaling pathways and multiple cytokines. Activation of protein kinase C (PKC) enhances the angiogenic process and is involved in the signaling of vascular endothelial growth factor (VEGF). We have demonstrated a dramatic increase in the angiogenic response to oxygen-induced retinal ischemia in transgenic mice overexpressing PKC beta 2 isoform and a significant decrease in retinal neovascularization in PKC beta isoform null mice. The mitogenic action of VEGF, a potent hypoxia-induced angiogenic factor, was increased by 2-fold in retinal endothelial cells by the overexpression of PKC beta 1 or beta 2 isoforms and inhibited significantly by the overexpression of a dominant-negative PKC beta 2 isoform but not by the expression of PKC alpha, delta, and zeta isoforms. Association of PKC beta 2 isoform with retinoblastoma protein was discovered in retinal endothelial cells, and PKC beta 2 isoform increased retinoblastoma phosphorylation under basal and VEGF-stimulated conditions. The potential functional consequences of PKC beta-induced retinoblastoma phosphorylation could include enhanced E2 promoter binding factor transcriptional activity and increased VEGF-induced endothelial cell proliferation.

    Funded by: NEI NIH HHS: EY10827, R01 EY5110

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;2;721-6

  • Phosphorylation of the cytoplasmic domain of the integrin CD18 chain by protein kinase C isoforms in leukocytes.

    Fagerholm S, Morrice N, Gahmberg CG and Cohen P

    MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom. susanna.fagerholm@helsinki.fi

    The CD11/CD18 (beta(2)) integrins are leukocyte-specific adhesion receptors, and their ability to bind ligands on other cells can be activated by extracellular stimuli. During cell activation, the CD18 chain is known to become phosphorylated on serine and functionally important threonine residues located in the intracellular C-terminal tail. Here, we identify catalytic domain fragments of protein kinase C (PKC) delta and PKCbetaI/II as the major protein kinases in leukocyte extracts that phosphorylate a peptide corresponding to the cytoplasmic tail of the integrin CD18 chain. The sites phosphorylated in vitro were identified as Ser-745 and Thr-758. PKCalpha and PKCeta also phosphorylated these residues, and PKCalpha additionally phosphorylated Thr-760. Ser-745, a novel site, was shown to become phosphorylated in T cells in response to phorbol ester stimulation. Ser-756, a residue not phosphorylated by PKC isoforms, also became phosphorylated in T cells after phorbol ester stimulation. When leukocyte extracts were subjected to affinity chromatography on agarose to which residues 751-761 of the CD18 chain phosphorylated at Thr-758 were bound covalently, the only proteins that bound specifically were identified as isoforms of 14-3-3 proteins. Thus, PKC-mediated phosphorylation of CD18 after cell stimulation could lead to the recruitment of 14-3-3 proteins to the activated integrin, which may play a role in regulating its adhesive state or ability to signal.

    The Journal of biological chemistry 2002;277;3;1728-38

  • The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta.

    Strack V, Krützfeldt J, Kellerer M, Ullrich A, Lammers R and Häring HU

    Medical Clinic, Department IV, Eberhard-Karls-University Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany.

    To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

    Biochemistry 2002;41;2;603-8

  • Protein kinase C beta isoenzymes in diabetic kidneys and their relation to nephroprotective actions of the ACE inhibitor lisinopril.

    Pfaff IL and Vallon V

    Department of Pharmacology, Faculty of Medicine, University of Tübingen, Germany.

    Inhibitors of angiotensin-converting enzyme (ACE) or beta isoforms of protein kinase C (PKC) are nephroprotective in diabetes mellitus. We investigated the influence of streptozotocin (STZ)-induced diabetes mellitus and of treatment with the ACE inhibitor lisinopril (4 mg/kg p.o. twice daily for 4 weeks) on the expression of PKC beta 1 and PKC beta 2 in the renal cortex of female Sprague-Dawley rats. Immunohistochemistry indicated an enhanced renocortical accumulation of macrophages expressing both MHC II, a marker for antigen-presenting cells, as well as PKC beta 2 in STZ diabetes which was confirmed by Western blotting demonstrating an enhanced renocortical expression of MHC II (1.8-fold) as well as of membrane-associated PKC beta 2 (1.9-fold). Whereas immunohistochemistry could not detect unequivocal alterations, Western blotting showed a rise in the renocortical expression of membrane-associated PKC beta 1 (1.7-fold) in STZ diabetes. Lisinopril lowered renocortical albumin content and proteinuria in STZ diabetes and attenuated the enhanced accumulation of macrophages expressing PKC beta 2 as well as the increase of membrane-associated expression of PKC beta 1 and PKC beta 2 in the renal cortex. The data suggest that the nephroprotective actions of the ACE inhibitor lisinopril in experimental diabetes mellitus were associated with and thus could be mediated in part by inhibition of diabetes-induced activation of PKC beta isoenzymes in the renal cortex.

    Kidney & blood pressure research 2002;25;5;329-40

  • Investigation of the inhibitory effects of chelerythrine chloride on the translocation of the protein kinase C betaI, betaII, zeta in human neutrophils.

    Siomboing X, Gressier B, Dine T, Brunet C, Luyckx M, Cazin M and Cazin JC

    Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Pharmacologie, Pharmacocinétique et Pharmacie Cliniques, Lille, France.

    The protein kinase C (PKC) is a serine/threonine kinase, consisting of different isoforms, implicated in numerous processes of signal transduction. To understand this enzyme well, different pharmacological tools were developed. To activate PKC specifically, phorbol esters were previously used but recent research has shown that these compounds are able to stimulate other proteins. Our model is the respiratory burst in the polymorphonuclear neutrophils. A decrease in the inflammatory process was measured using chelerythrine chloride. Action on PKC was proved by a binding study and by showing the absence of translocation of this enzyme from the cytoplasm to the plasmic membrane during stimulation.

    Farmaco (Società chimica italiana : 1989) 2001;56;11;859-65

  • Protein kinase C regulates the phosphorylation and cellular localization of occludin.

    Andreeva AY, Krause E, Müller EC, Blasig IE and Utepbergenov DI

    Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin-Buch and Charité, Humboldt Universität Berlin, 13092 Berlin, Germany.

    Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.

    The Journal of biological chemistry 2001;276;42;38480-6

  • PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization.

    Kang SW, Wahl MI, Chu J, Kitaura J, Kawakami Y, Kato RM, Tabuchi R, Tarakhovsky A, Kawakami T, Turck CW, Witte ON and Rawlings DJ

    Department of Pediatrics, University of California, Los Angeles, CA 90095-1752, USA.

    Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.

    Funded by: NICHD NIH HHS: HD37091, R01 HD037091

    The EMBO journal 2001;20;20;5692-702

  • HIV envelope gp120 activates human arterial smooth muscle cells.

    Schecter AD, Berman AB, Yi L, Mosoian A, McManus CM, Berman JW, Klotman ME and Taubman MB

    Zena and Michael A. Wiener Cardiovascular Institute and Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, NY 10029, USA. alison.schecter@.mssm.edu

    There have been increasing reports of acute coronary thrombotic events in patients with HIV. Although these clinical events have been attributed primarily to dyslipidemia associated with protease inhibitor therapy, autopsy studies in children with HIV suggest the presence of an underlying arteriopathy. This study demonstrates that the HIV envelope protein, gp120, activates human arterial smooth muscle cells to express tissue factor, the initiator of the coagulation cascade. The induction of tissue factor by gp120 is mediated by two biologically relevant coreceptors for HIV infection, CXCR4 and CCR5, and is also dependent on the presence of functional CD4. Induction of tissue factor by gp120 requires activation of mitogen-activating protein kinases, activation of protein kinase C, and generation of reactive oxygen species, signaling pathways that have protean effects on smooth muscle cell physiology. The activation of smooth muscle cells by gp120 may play an important role in the vascular, thrombotic, and inflammatory responses to HIV infection.

    Funded by: NHLBI NIH HHS: HL03801, HL29019, P01 HL029019; NIDDK NIH HHS: P01DKJ6492; NIMH NIH HHS: MH52974

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;18;10142-7

  • Glycogen synthase kinase-3: properties, functions, and regulation.

    Ali A, Hoeflich KP and Woodgett JR

    Division of Experimental Therapeutics, Ontario Cancer Institute, 610-University Avenue, Toronto, Ontario, Canada M5G 2M9. aali@uwindsor.ca

    Chemical reviews 2001;101;8;2527-40

  • Insulin regulates alternative splicing of protein kinase C beta II through a phosphatidylinositol 3-kinase-dependent pathway involving the nuclear serine/arginine-rich splicing factor, SRp40, in skeletal muscle cells.

    Patel NA, Chalfant CE, Watson JE, Wyatt JR, Dean NM, Eichler DC and Cooper DR

    Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612, USA.

    Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.

    The Journal of biological chemistry 2001;276;25;22648-54

  • Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    Chen L, Haider K, Ponda M, Cariappa A, Rowitch D and Pillai S

    Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts 02129, USA.

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

    Funded by: NCI NIH HHS: CA 69618; NIAID NIH HHS: AI 33507

    The Journal of biological chemistry 2001;276;24;21737-44

  • The carboxyl terminus of protein kinase c provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1.

    Gao T, Toker A and Newton AC

    Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640, USA.

    The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C betaII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.

    Funded by: NCI NIH HHS: CA75134; NIDDK NIH HHS: P01 DK54441

    The Journal of biological chemistry 2001;276;22;19588-96

  • Evidence for direct protein kinase-C mediated modulation of N-methyl-D-aspartate receptor current.

    Liao GY, Wagner DA, Hsu MH and Leonard JP

    Laboratory of Integrative Neuroscience, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.

    Protein kinase-C (PKC) activation differentially affects currents from N-methyl-D-aspartate (NMDA) type glutamate receptors depending upon their subunit composition. Experiments using chimeras initially indicated that the cytoplasmic C-terminal tails of NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits contain the amino acid residues responsible for the observed disparity of PKC effects. However, truncation and point mutation experiments have suggested that PKC action on NMDA receptors may be entirely indirect, working via the phosphorylation of associated proteins. Here we suggest that PKC does, in fact, affect NR2B/NR1-011 NMDA currents by direct phosphorylation of the NR2B tail at residues S1303 and S1323. Replacement of either of these residues with Ala severely reduces PKC potentiation. To verify that S1303 and S1323 are sites of direct phosphorylation by PKC, synthetic peptides from the regions surrounding these sites were used as substrates for in vitro assays with purified rat brain PKC. These results indicate that PKC can directly phosphorylate S1303 and S1323 in the NR2B C terminus, leading to enhanced currents through NMDA receptor channels. The direct action of PKC on certain NMDA receptor subtypes may be important in any physiological or pathological process where PKC and NR2B/NR1 receptors interact.

    Funded by: NINDS NIH HHS: R01-NS31962-02

    Molecular pharmacology 2001;59;5;960-4

  • Regulation of the ErbB3 binding protein Ebp1 by protein kinase C.

    Lessor TJ and Hamburger AW

    Molecular and Cellular Biology Program, University of Maryland, Baltimore, MD 21201, USA.

    Ebp1, a 47 kDa ubiquituously expressed protein, binds the ErbB3 receptor in human serum starved breast cancer cell lines and dissociates from ErbB3 on treatment with the ErbB3 ligand, Heregulin (HRG). However, the mechanism of Ebp1-ErbB3 association/dissociation is not understood. Since Ebp1 contains six putative Protein Kinase C serine/threonine phosphorylation sites, we examined the ability of PKC to phosphorylate Ebp1 and to regulate Ebp1-ErbB3 binding. We found that Ebp1 was basally phosphorylated in AU565 breast cancer cells on serine/threonine residues and that this phosphorylation was enhanced by heregulin treatment. Both serine and threonine residues of a GST-Ebp1 fusion protein were phosphorylated by PKC in vitro. In vivo, we demonstrated that basal Ebp1 phosphorylation was dependent upon PKC. However, HRG-induced phosphorylation of Ebp1 occurred predominantly in a PKC-independent manner. The ability of Ebp1 to associate with ErbB3 in serum-starved NIH3T3 cells overexpresssing ErbB3 was abrogated by treating cells with a PKC inhibitor. These findings suggest that PKC plays a role in regulating phosphorylation and function of Ebp1 in vivo.

    Funded by: NCI NIH HHS: R01 CA76047

    Molecular and cellular endocrinology 2001;175;1-2;185-91

  • Activation of annexin 7 by protein kinase C in vitro and in vivo.

    Caohuy H and Pollard HB

    Department of Anatomy and Cell Biology, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.

    Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.

    The Journal of biological chemistry 2001;276;16;12813-21

  • Roles for beta II-protein kinase C and RACK1 in positive and negative signaling for superoxide anion generation in differentiated HL60 cells.

    Korchak HM and Kilpatrick LE

    Department of Pediatrics, University of Pennsylvania School of Medicine, The Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA. korchak@email.chop.edu

    beta-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O(2)). Neutrophils and neutrophilic HL60 cells contain both betaI and betaII-PKC, isotypes that are derived by alternate splicing. betaI-PKC-positive and betaI-PKC null HL60 cells generated equivalent amounts of O(2) in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of betaII-PKC from betaI-PKC null cells inhibited ligand-initiated O(2) generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47(phox), with betaII-PKC but not with RACK1, a binding protein for betaII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of beta-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O(2) generation. Therefore, betaII-PKC but not betaI-PKC is essential for activation of O(2) generation and plays a positive role in signaling for NADPH oxidase activation in association with p47(phox). In contrast, RACK1 is involved in negative signaling for O(2) generation. RACK1 binds to betaII-PKC but not with the p47(phox).betaII-PKC complex. RACK1 may divert betaII-PKC to other signaling pathways requiring beta-PKC for signal transduction. Alternatively, RACK1 may sequester betaII-PKC to down-regulate O(2) generation.

    Funded by: NIAID NIH HHS: AI24840

    The Journal of biological chemistry 2001;276;12;8910-7

  • Essential activation of PKC-delta in opioid-initiated cardioprotection.

    Fryer RM, Wang Y, Hsu AK and Gross GJ

    Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

    Stimulation of the delta(1)-opioid receptor confers cardioprotection to the ischemic myocardium. We examined the role of protein kinase C (PKC) after delta-opioid receptor stimulation with TAN-67 or D-Ala(2)-D-Leu(5)-enkephalin (DADLE) in a rat model of myocardial infarction induced by a 30-min coronary artery occlusion and 2-h reperfusion. Infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of the area at risk (IS/AAR). Control animals, subjected to ischemia and reperfusion, had an IS/AAR of 59.9 +/- 1.8. DADLE and TAN-67 administered before ischemia significantly reduced IS/AAR (36.9 +/- 3.9 and 36.7 +/- 4.7, respectively). The delta(1)-selective opioid antagonist 7-benzylidenenaltrexone (BNTX) abolished TAN-67-induced cardioprotection (54.4 +/- 1.3). Treatment with the PKC antagonist chelerythrine completely abolished DADLE- (61.8 +/- 3.2) and TAN-67-induced cardioprotection (55.4 +/- 4.0). Similarly, the PKC antagonist GF 109203X completely abolished TAN-67-induced cardioprotection (54.6 +/- 6.6). Immunofluorescent staining with antibodies directed against specific PKC isoforms was performed in myocardial biopsies obtained after 15 min of treatment with saline, chelerythrine, BNTX, or TAN-67 and chelerythrine or BNTX in the presence of TAN-67. TAN-67 induced the translocation of PKC-alpha to the sarcolemma, PKC-beta(1) to the nucleus, PKC-delta to the mitochondria, and PKC-epsilon to the intercalated disk and mitochondria. PKC translocation was abolished by chelerythrine and BNTX in TAN-67-treated rats. To more closely examine the role of these isoforms in cardioprotection, we utilized the PKC-delta selective antagonist rottlerin. Rottlerin abolished opioid-induced cardioprotection (48.9 +/- 4.8) and PKC-delta translocation without affecting the translocation of PKC-alpha, -beta(1), or -epsilon. These results suggest that PKC-delta is a key second messenger in the cardioprotective effects of delta(1)-opioid receptor stimulation in rats.

    Funded by: NHLBI NIH HHS: HL-08311

    American journal of physiology. Heart and circulatory physiology 2001;280;3;H1346-53

  • Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling.

    Cunningham ML, Waldo GL, Hollinger S, Hepler JR and Harden TK

    Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7365 and the Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322-3090.

    RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTPgammaS-stimulated Galpha(11) signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10-20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-betat activity than of GTPgammaS-promoted PLC-betat activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4beta-phorbol 12beta-myristate 13alpha-acetate and, to a lesser extent, by the P2Y(2) receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTPgammaS-stimulated PLC-betat activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y(1) receptor and Galpha(q)betagamma. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.

    Funded by: NIGMS NIH HHS: GM29536, GM38213

    The Journal of biological chemistry 2001;276;8;5438-44

  • HIV-1 Tat promotes monocyte chemoattractant protein-1 secretion followed by transmigration of monocytes.

    Park IW, Wang JF and Groopman JE

    Division of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA 02115, USA.

    The mechanism whereby HIV-infected cells transit from the bloodstream into tissues is not well defined. This phenomenon was addressed by studying the effects of HIV-1 Tat, a protein secreted by infected cells, on human lung microvascular endothelial cells (HMVEC-Ls). It was found that monocyte chemoattractant protein-1 (MCP-1) was released from HMVEC-Ls in a dose- and time-dependent manner after Tat treatment. MCP-1 is a potent beta-chemokine that recruits monocytes and T cells and promotes cell adhesion and transmigration across an endothelial monolayer. It was also observed that MCP-1 and the culture medium from Tat-treated HMVEC-Ls were chemotactic for CD14(+) monocytes from human peripheral blood and for THP-1, a promonocytic cell line used as a model system. To characterize the signaling pathways underlying the observed induction of MCP-1, HMVEC-Ls were treated with 2 different protein kinase inhibitors: PD98059, a MAP kinase inhibitor, and GF109203X, a protein kinase C (PKC) inhibitor. MCP-1 release was significantly reduced when PKC was inhibited, and slightly decreased when PI3 kinase was blocked; no effect on MCP-1 release was observed on MAP kinase inhibition. Similarly, transmigration of THP-1 cells was significantly impaired by the PKC inhibitor, but not by the other tested inhibitors. These data indicate that the HIV-1 Tat protein may act as a protocytokine by causing the release of MCP-1 from the endothelial monolayer, and thereby facilitating monocyte transmigration into tissues via a PKC signaling pathway.

    Funded by: NHLBI NIH HHS: HL53745, HL61940

    Blood 2001;97;2;352-8

  • Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst.

    Dang PM, Fontayne A, Hakim J, El Benna J and Périanin A

    Institut National de la Santé et de la Recherche Médicale Unité 479, Centre Hospitalier Universitaire Xavier Bichat, Paris, France.

    Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.

    Journal of immunology (Baltimore, Md. : 1950) 2001;166;2;1206-13

  • Role of two conserved cytoplasmic threonine residues (T410 and T412) in CD5 signaling.

    Vilà JM, Calvo J, Places L, Padilla O, Arman M, Gimferrer I, Aussel C, Vives J and Lozano F

    Servei d'Immunologia, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clínic, Barcelona, Spain.

    CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.

    Journal of immunology (Baltimore, Md. : 1950) 2001;166;1;396-402

  • [HIV-1 Tat protein induces IL-10 production by human monocytes: implications of the PKC and calcium pathway].

    Bennasser Y, Yamina B, Contreras X, Xavier C, Moreau M, Marc M, Le Clerc C, Catherine L, Badou A, Abdallah B and Bahraoui E

    Laboratoire d'Immuno-Virologie, Université Paul Sabatier 118, route de Narbonne, 31062 Toulouse.

    In asymptomatic patients infected by HIV-1, the level of IL-10, a cytokine with immunosuppressive activity, is associated with the course of HIV infection towards AIDS. We show that HIV-1 Tat, a viral protein secreted by infected cells, induces IL-10 production by human peripheral blood monocytes. The analysis of the signal transduction pathways strongly suggests that the protein kinase C may play an essential role in this induction. Stimulation by Tat induces nuclear translocation of the transcription factor NFkB the activation of which seems to be necessary for IL-10 production. Using microspectrofluorimetry and confocal microscopy, we also show that Tat induces a calcium influx.

    Journal de la Societe de biologie 2001;195;3;319-26

  • Involvement of protein kinase C in HIV-1 gp120-induced apoptosis in primary endothelium.

    Huang MB and Bond VC

    Department of Biochemistry, Morehouse School of Medicine, Atlanta, Georgia 30310-1495, USA.

    We previously showed that HIV-1 gp120-induced apoptosis in primary human umbilical vein endothelial cell cultures (HUVEC), through CCR5 and CXCR4. Here, we have found that agonists of protein kinase C (PKC), basic fibroblast growth factor (bFGF), and short exposure to low concentrations of phorbol esters were found to block gp120-induced apoptosis in HUVEC cultures. PKC antagonists, sphingosine, H7, and extended exposure of cultures to high concentrations of phorbol esters were also found to block gp120-induced apoptosis in HUVEC cultures. A significant increase in the total amount of cellular PKC enzymatic activity was observed on exposure of HUVEC to gp120. No increase in total PKC activity was observed on exposure of HUVECs to the natural ligands SDF-1alpha, or regulated-on-activation normal T-expressed and secreted (RANTES) cells, and gp120-induced PKC induction was found to be totally blocked by CXCR4 antibodies and partially blocked by the caspase 3 inhibitor, DEVD-CHO. Alternatively, CXCR4 antibodies and DEVD-CHO totally blocked apoptosis. Finally, gp120-induced effects were found to be insensitive to pertussis toxin. Accumulated evidence suggests PKC involvement at multiple points in the gp120-induced apoptotic pathway; also suggests involvement of the CXCR4 receptor internalization pathway, and potentially suggests different downstream effects of gp120-receptor interactions and natural ligand-receptor interactions.

    Funded by: NCRR NIH HHS: G12-RR03034

    Journal of acquired immune deficiency syndromes (1999) 2000;25;5;375-89

  • Expression of protein kinase C isozymes in nonpregnant and pregnant human myometrium.

    Hurd WW, Fomin VP, Natarajan V, Brown HL, Bigsby RM and Singh DM

    Department of Obstetrics and Gynecology and the Department of Internal Medicine, Indiana University School of Medicine, Indianapolis, USA.

    Objective: The aim of this study was to compare the distributions of protein kinase C isozymes in human nonpregnant and pregnant myometrial tissues and primary cell cultures.

    Myometrial tissues were obtained at hysterectomy from nonpregnant women and at cesarean delivery from women both before and during early labor at term. Western immunoblot analysis was performed on homogenates of myometrial tissues and primary cell cultures with monoclonal antibodies specific for protein kinase C isozymes. Redistribution and translocation of protein kinase C were examined by means of immunocytochemical methods.

    Results: Nonpregnant myometrial tissues contained protein kinase C isozymes alpha, gamma, delta, mu, iota, and zeta but not beta(1), beta(2), theta, or epsilon. Pregnant myometrial tissues both before and during early labor contained the same protein kinase C isozymes and also beta(1) and beta(2). The protein kinase C isozyme distribution in primary myometrial cell cultures was identical to that in the myometrial tissues. Protein kinase C redistribution and translocation were demonstrated in these cultured myometrial cells.

    Conclusion: Both human myometrial tissues and primary cell cultures expressed a broad range of protein kinase C isozymes. Protein kinase C isozymes beta(1) and beta(2) were absent in nonpregnant myometrium but were induced during pregnancy. Labor at term did not alter protein kinase C isozyme expression.

    Funded by: NICHD NIH HHS: HD36692

    American journal of obstetrics and gynecology 2000;183;6;1525-31

  • Tat protein of human immunodeficiency virus type 1 induces interleukin-10 in human peripheral blood monocytes: implication of protein kinase C-dependent pathway.

    Badou A, Bennasser Y, Moreau M, Leclerc C, Benkirane M and Bahraoui E

    Laboratoire d'Immuno-Virologie EA3038, Université Paul Sabatier, 31062 Toulouse Cedex, France.

    The clinical manifestations observed in human immunodeficiency virus type 1 (HIV-1)-infected patients are primarily due to the capacity of the virus and its components to inactivate the immune system. HIV-1 Tat protein could participate in this immune system disorder. This protein is secreted by infected cells of HIV-infected patients and is free in the plasma, where it can interact and be taken up by both infected and noninfected cells. In asymptomatic patients infected by HIV-1, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with disease progression to AIDS. In the present work, we tested the capacity of Tat to induce IL-10 production by peripheral blood monocytes of healthy donors. The results show that Tat causes the production of IL-10 in a dose- and stimulation time-dependent manner. Investigations of the mechanisms involved in signal transduction show that (i) the calcium pathway is not or only slightly involved in Tat-induced IL-10 production, (ii) the protein kinase C pathway plays an essential role, and (iii) monocyte stimulation by Tat results in the intranuclear translocation of transcription factor NF-kappaB and in the induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2; activation of these two potential substrates of protein kinase C is required for the production of IL-10. Finally, our results suggest that the effect of Tat is exerted at the membrane level and that the active domain is located within N-terminal residues 1 to 45. This production of IL-10 induced by Tat could participate in the progression of HIV infection to AIDS.

    Journal of virology 2000;74;22;10551-62

  • Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A.

    Fang X, Yu SX, Lu Y, Bast RC, Woodgett JR and Mills GB

    Department of Molecular Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

    Glycogen synthase kinase 3 (GSK-3) is implicated in multiple biological processes including metabolism, gene expression, cell fate determination, proliferation, and survival. GSK-3 activity is inhibited through phosphorylation of serine 21 in GSK-3 alpha and serine 9 in GSK-3 beta. These serine residues of GSK-3 have been previously identified as targets of protein kinase B (PKB/Akt), a serine/threonine kinase located downstream of phosphatidylinositol 3-kinase. Here, we show that serine 21 in GSK-3 alpha and serine 9 in GSK-3 beta are also physiological substrates of cAMP-dependent protein kinase A. Protein kinase A physically associates with, phosphorylates, and inactivates both isoforms of GSK-3. The results indicate that depending on the stimulatory context, the activity of GSK-3 can be modulated either by growth factors that work through the phosphatidylinositol 3-kinase-protein kinase B cascade or by hormonal stimulation of G protein-coupled receptors that link to changes in intracellular cAMP levels.

    Funded by: NCI NIH HHS: CA64602, CA82716, P01 CA064602, R01 CA082716

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;22;11960-5

  • Identification and characterization of PRKCBP1, a candidate RACK-like protein.

    Fossey SC, Kuroda S, Price JA, Pendleton JK, Freedman BI and Bowden DW

    Molecular Genetics Program, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA.

    The receptors for activated C-kinase (RACK) family of proteins function as anchors for activated protein kinase C (PKC) isoenzymes. Using a monoclonal antibody to RACK1 in the screening of a human hippocampus cDNA library, we identified a novel member of the RACK family, designated PRKCBP1. Immunoprecipitation assays performed with a GST-fused PRKCBP1 protein suggest the carboxy terminus of PRKCBP1 interacts specifically with PKCbetaI. Northern analysis detected a 3.1-kb PRKCBP1 transcript in all tissues examined including brain, heart, liver, lung, pancreas, skeletal muscle, kidney, and spleen. The PRKCBP1 gene has been localized to human Chromosome (Chr) 20q12-13.1. Several groups have reported evidence for genetic linkage of Type II diabetes to this region in Caucasian families. This localization, combined with the observation that abnormalities in the activation, translocation, and inhibition of PKC occur in the development of Type II diabetes, suggested that PRKCBP1 was a candidate for contributing to Type II diabetes. We determined the PRKCBP1 coding sequence is 1845 bp in length, dispersed over 9 exons, spanning approximately 34 kb of genomic DNA. SSCP analysis was used to screen PRKCBP1 coding regions for mutations or polymorphisms in 100 Caucasian Type II diabetics and 100 Caucasian unaffected individuals. A silent C/T transition (bp1413, Thr137) was present in 23% of both diabetic and control individuals. A C/T transition (bp198) was also identified in a single diabetic individual, which resulted in no coding change (Ser66). The results of this analysis suggest that PRKCBP1 coding variations are uncommon and do not contribute to Type II diabetes in the general population.

    Funded by: NIDDK NIH HHS: R01 DK41269; PHS HHS: R01 47480

    Mammalian genome : official journal of the International Mammalian Genome Society 2000;11;10;919-25

  • Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets.

    Chung SH, Polgar J and Reed GL

    Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, MA 02115, USA.

    We postulated that the syntaxins, because of their key role in SNARE complex formation and exocytosis, could be important targets for signaling by intracellular kinases involved in secretion. We found that syntaxin 4 was phosphorylated in human platelets treated with a physiologic agent that induces secretion (thrombin) but not when they were treated with an agent that prevents secretion (prostacyclin). Syntaxin 4 phosphorylation was blocked by inhibitors of activated protein kinase C (PKC), and, in parallel assays, PKC inhibitors also blocked secretion from thrombin-activated platelets. In platelets, cellular activation by thrombin or phorbol 12-myristate 13-acetate decreased the binding of syntaxin 4 with SNAP-23, another platelet t-SNARE. Phosphatase inhibitors increased syntaxin 4 phosphorylation and further decreased syntaxin 4-SNAP-23 binding induced by cell activation. Conversely, a PKC inhibitor blocked syntaxin 4 phosphorylation and returned binding of syntaxin 4-SNAP-23 to that seen in nonstimulated platelets. In vitro, PKC directly phosphorylated platelet syntaxin 4 and recombinant syntaxin 4. PKC phosphorylation in vitro inhibited (71 +/- 8%) the binding of syntaxin 4 to SNAP-23. These results provide evidence that extracellular activation can be coupled through intracellular PKC signaling so as to modulate SNARE protein interactions involved in platelet exocytosis.

    Funded by: NHLBI NIH HHS: HL-64057

    The Journal of biological chemistry 2000;275;33;25286-91

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.

    Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG and Lau AF

    Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. plampe@fhcrc.org

    Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

    Funded by: NCI NIH HHS: R01 CA052098; NHLBI NIH HHS: HL58732, R01 HL058732; NIGMS NIH HHS: GM46277, GM55632, R01 GM046277, R01 GM055632

    The Journal of cell biology 2000;149;7;1503-12

  • Release of calcium from inositol 1,4,5-trisphosphate receptor-regulated stores by HIV-1 Tat regulates TNF-alpha production in human macrophages.

    Mayne M, Holden CP, Nath A and Geiger JD

    Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada.

    HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-alpha, a cytopathic cytokine linked to the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-alpha production in macrophages, and we have shown that Tat releases calcium from inositol 1,4, 5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-alpha production was dependent on the release of intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-dependently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2 inhibitors. Xestospongin C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-alpha production. These results demonstrate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconventional PKC isoforms play an important role in Tat-induced TNF-alpha production.

    Journal of immunology (Baltimore, Md. : 1950) 2000;164;12;6538-42

  • Regulation of receptor-mediated protein kinase C membrane trafficking by autophosphorylation.

    Feng X, Becker KP, Stribling SD, Peters KG and Hannun YA

    Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. In response to activation of cell-surface receptors, PKC is directed to the plasma membrane by two membrane-targeting domains, namely the C1 and C2 regions. This is followed by the return of the enzyme to the cytoplasm, a process shown recently to require PKC autophosphorylation (Feng, X., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 26870-26874). In the present study, we examined mechanisms of translocation and reverse translocation and the role of autophosphorylation in these processes. By visualizing the trafficking of wild-type as well as mutant PKCbetaII in live cells, we demonstrated that in response to cell-surface receptor activation, the function of the C1 region is required but not sufficient for recruitment of the enzyme to the plasma membrane. The C2 region is also critical in anchoring the enzyme to the plasma membrane. Furthermore, the inability of a kinase-deficient PKC to undergo reverse translocation was restored by the addition of intracellular calcium chelators, suggesting a role for the C2 region in the persistent phase of translocation. On the other hand, the inability of a C2 deletion mutant (C1 region intact) to translocate in response to agonist was reversed in mutants lacking kinase activity or by mutation of the Ser(660) autophosphorylation site to alanine, suggesting that autophosphorylation of this site is required for opposing the action of the C2 region. Therefore, the membrane-targeting function of the C1 region is facilitated by the C2 region and appears to be opposed by autophosphorylation. Taken together, these findings provide novel evidence of the functional regulation of reversible PKC membrane localization by autophosphorylation, and they show that the dynamic translocation of PKC in response to agonists is tightly regulated in a collaborative fashion by the C1 and C2 regions in balance with the effects of autophosphorylation.

    Funded by: NHLBI NIH HHS: HL-43707

    The Journal of biological chemistry 2000;275;22;17024-34

  • HIV-1 reverse transcriptase is phosphorylated in vitro and in a cellular system.

    Idriss H, Kawa S, Damuni Z, Thompson EB and Wilson SH

    Sealy Center for Molecular Science, The University of Texas Medical Branch Galveston, TX 77555-0851, USA. hi@st-and.ac.uk

    Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases. The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus. HIV-1 RT has several potential sites for phosphorylation that could regulate its activities. In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism. Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases. Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT. Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo. However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation. Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.

    Funded by: NIDDK NIH HHS: DK41058

    The international journal of biochemistry & cell biology 1999;31;12;1443-52

  • The role of DOC-2/DAB2 protein phosphorylation in the inhibition of AP-1 activity. An underlying mechanism of its tumor-suppressive function in prostate cancer.

    Tseng CP, Ely BD, Pong RC, Wang Z, Zhou J and Hsieh JT

    Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9110, USA.

    DOC-2/DAB2, a novel phosphoprotein with signal-transducing capability, inhibits human prostatic cancer cells (Tseng, C.-P., Ely, B. D., Li, Y., Pong, R.-C., and Hsieh, J.-T. (1998) Endocrinology 139, 3542-3553). However, its mechanism of action is not understood completely. This study delineates the functional significance of DOC-2/DAB2 protein phosphorylation and demonstrates that in vivo activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces DOC-2/DAB2 phosphorylation, including a serine residue at position 24. Mutation of Ser(24) to Ala reduced DOC-2/DAB2 phosphorylation by PKC. Using a synthetic Ser(24) peptide (APS(24)KKEKKKGSEKTD) or recombinant DOC-2/DAB2 as substrates, PKCbetaII, PKCgamma, and PKCdelta (but not casein kinase II) directly phosphorylated Ser(24) in vitro. This indicates that DOC-2/DAB2 is a PKC-specific substrate. Since expression of wild-type DOC-2/DAB2, but not the S24A mutant, inhibited TPA-induced AP-1 activity in prostatic epithelial cells, phosphorylation of Ser(24) appears to play a critical role in modulating TPA-induced AP-1 activity. Taken together, these data suggest that PKC-regulated phosphorylation of DOC-2/DAB2 protein may help its growth inhibitory function.

    Funded by: NCI NIH HHS: CA59939

    The Journal of biological chemistry 1999;274;45;31981-6

  • Regulation of ARNO nucleotide exchange by a PH domain electrostatic switch.

    Santy LC, Frank SR, Hatfield JC and Casanova JE

    Combined Program in Pediatric Gastroenterology and Nutrition Massachusetts General Hospital East, Charlestown, Massachusetts 02129, USA.

    ARNO is a member of a family of guanine nucleotide exchange factors that activate small GTPases called ADP-ribosylation factors (ARFs) [1] [2] [3], which regulate vesicular trafficking and, in one case (ARF6), also regulate cortical actin structure [4]. ARNO is located at the plasma membrane, and in the presence of activated protein kinase C (PKC) can induce cortical actin rearrangements reminiscent of those produced by active ARF6 [5] [6] [7] [8]. High-affinity binding of ARNO to membranes, which is required for exchange activity, is mediated cooperatively by a pleckstrin homology (PH) domain and an adjacent carboxy-terminal polybasic domain [3] [9]. ARNO is phosphorylated in vivo by PKC on a single serine residue, S392, located within the carboxy-terminal polybasic domain. Mutation of S392 to alanine does not prevent ARNO-mediated actin rearrangements, suggesting that phosphorylation does not lead to ARNO activation [6]. Here, we report that phosphorylation negatively regulates ARNO exchange activity through a 'PH domain electrostatic switch'. Introduction of a negatively charged phosphate into the polybasic domain reduced interaction of ARNO with membranes both in vitro and in vivo, and inhibited exchange in vitro. This regulated membrane association is similar to the myristoyl electrostatic switch that controls membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) [10], but to our knowledge is the first demonstration of an electrostatic switch regulating the membrane interaction of a protein containing a PH domain. This mechanism allows regulation of ARNO lipid binding and exchange activity at two levels, phosphoinositide-dependent recruitment and PKC-dependent displacement from the membrane.

    Funded by: NIAID NIH HHS: AI-32991; NIDDK NIH HHS: F32-DK-09924

    Current biology : CB 1999;9;20;1173-6

  • Biochemical characterization of pKi67 with the identification of a mitotic-specific form associated with hyperphosphorylation and altered DNA binding.

    MacCallum DE and Hall PA

    Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, United Kingdom.

    Although widely used as an operational marker of proliferation, the cell cycle-regulated Ki67 protein is of unknown function. pKi67 is found predominantly in the nucleolus in cycling interphase cells and moves to become perichromosomal during mitosis. We have performed a detailed immunochemical analysis of pKi67 in HeLa cells and report the existence of a novel hyperphosphorylated form in mitosis. Two isoforms can be identified on immunoblots as a consequence of the previously described alternative splicing. In extracts from mitotic cells both these isoforms have considerably reduced mobility. Treatment with phosphatase converts the mitotic form to the interphase form. Immunoprecipitated pKi67 can be phosphorylated in vitro both by cdc2/cyclin B and by protein kinase C, and treatment by PKC leads to the full mobility shift. Treatment of nocodazole-arrested mitotic HeLa cells with staurosporine causes a dephosphorylation of pKi67 to the interphase state and a concomitant change in the localization of pKi67 with movement away from the perichromosomal layer to cytoplasmic dots that colocalize with nucleolin. These data indicate that pKi67 localization is regulated by the action of cell cycle-specific kinase(s) and phosphatase(s). The data presented here provide a starting point for the analysis of pKi67 function and regulation.

    Experimental cell research 1999;252;1;186-98

  • Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates.

    Tözsér J, Bagossi P, Boross P, Louis JM, Majerova E, Oroszlan S and Copeland TD

    Department of Biochemistry and Molecular Biology, University Medical School of Debrecen, Hungary. tozser@indi.biochem.dote.hu

    Vimentin, a cellular substrate of HIV type 1 (HIV-1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser-phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C-terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser-phosphorylated peptide was not hydrolyzed by the enzyme at the Ser-Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser-phosphorylated and Tyr-phosphorylated forms of model substrates were also tested as substrates of the HIV-1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV-1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus-host cell interactions.

    European journal of biochemistry 1999;265;1;423-9

  • Coordinated movement of RACK1 with activated betaIIPKC.

    Ron D, Jiang Z, Yao L, Vagts A, Diamond I and Gordon A

    Department of Neurology, University of California, San Francisco, California 94110-3518, USA. dorit@itsa.ucsf.edu

    Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated betaIIPKC, determines the localization and functional activity of betaIIPKC. However, the mechanism by which RACK1 localizes activated betaIIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in the in situ association and movement of RACK1 and betaIIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and betaIIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves betaIIPKC from one intracellular site to another.

    Funded by: NIAAA NIH HHS: AA10039

    The Journal of biological chemistry 1999;274;38;27039-46

  • Genome duplications and other features in 12 Mb of DNA sequence from human chromosome 16p and 16q.

    Loftus BJ, Kim UJ, Sneddon VP, Kalush F, Brandon R, Fuhrmann J, Mason T, Crosby ML, Barnstead M, Cronin L, Deslattes Mays A, Cao Y, Xu RX, Kang HL, Mitchell S, Eichler EE, Harris PC, Venter JC and Adams MD

    The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA.

    Several publicly funded large-scale sequencing efforts have been initiated with the goal of completing the first reference human genome sequence by the year 2005. Here we present the results of analysis of 11.8 Mb of genomic sequence from chromosome 16. The apparent gene density varies throughout the region, but the number of genes predicted (84) suggests that this is a gene-poor region. This result may also suggest that the total number of human genes is likely to be at the lower end of published estimates. One of the most interesting aspects of this region of the genome is the presence of highly homologous, recently duplicated tracts of sequence distributed throughout the p-arm. Such duplications have implications for mapping and gene analysis as well as the predisposition to recurrent chromosomal structural rearrangements associated with genetic disease.

    Funded by: NHGRI NIH HHS: R01-HG01464

    Genomics 1999;60;3;295-308

  • Association of RACK1 and PKCbeta with the common beta-chain of the IL-5/IL-3/GM-CSF receptor.

    Geijsen N, Spaargaren M, Raaijmakers JA, Lammers JW, Koenderman L and Coffer PJ

    Department of Pulmonary Diseases, University Hospital Utrecht, Heidelberglaan 100, 3508 GA Utrecht, The Netherlands.

    Granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5 belong to a family of cytokines that regulate proliferation, differentiation and function of haematopoietic cells. Their receptor consists of a ligand specific alpha-chain and a signal transducing beta-chain (betac). While, the role of phosphotyrosine residues in the betac as mediators of downstream signalling cascades has been established, little is known about non-phosphotyrosine mediated events. To identify proteins interacting with betac, we screened a yeast two-hybrid library with the intracellular domain of betac. We found that RACK1, a molecule associating with activated PKC, PLCgamma and Src kinases, associated with the membrane proximal region of betac in both yeast two-hybrid, immunoprecipitation and GST-pull-down assays. The association of RACK1 was constitutive, demonstrating no alteration upon cellular stimulation. Furthermore, upon stimulation of cells with IL-5 or PMA, a complex of betac and PKCbeta was found. Together, these findings suggest a novel role for RACK1 as a possible adapter molecule associating with the intracellular domain of cytokine receptors.

    Oncogene 1999;18;36;5126-30

  • Protein kinase C delta associates with and phosphorylates Stat3 in an interleukin-6-dependent manner.

    Jain N, Zhang T, Kee WH, Li W and Cao X

    Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Dr., Singapore 117609.

    Stat3 is activated by phosphorylation on Tyr-705, which leads to dimer formation, nuclear translocation, and regulation of gene expression. Serine phosphorylation of Stat3 by mitogen-activated protein kinase has also been observed in cells responding to epidermal growth factor and shown to affect its tyrosine phosphorylation and transcriptional activity. Serine phosphorylation of Stat3 is also induced by interleukin-6 (IL-6) stimulation, which is shown to be independent of mitogen-activated protein kinase and sensitive to the Ser/Thr kinase inhibitor H7. In this study, we investigated whether protein kinase C (PKC) is the kinase that is induced and responsible for Stat3 serine phosphorylation by IL-6 stimulation and which isoform of PKCs is likely to be involved. Here, we report that Stat3 was specifically associated with PKC delta in vivo in an IL-6-dependent manner in several cell types. Furthermore, Stat3 was phosphorylated by PKC delta in vivo on Ser-727, which could be inhibited either by a specific PKC delta inhibitor or by a dominant-negative mutant of PKC delta. Finally, we showed that the phosphorylation of Stat3 by PKC delta led to a negative regulation of Stat3 DNA binding and transcriptional activity. These results indicate that PKC delta is likely to be the kinase that phosphorylates Stat3 in response to IL-6 stimulation and suggest a possible regulatory role of PKC delta on Stat3 function.

    The Journal of biological chemistry 1999;274;34;24392-400

  • HIV-1 Nef alters the expression of betaII and epsilon isoforms of protein kinase C and the activation of the long terminal repeat promoter in human astrocytoma cells.

    Ambrosini E, Slepko N, Kohleisen B, Shumay E, Erfle V, Aloisi F and Levi G

    Istituto Superiore di Sanità, Rome, Italy.

    In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.

    Glia 1999;27;2;143-51

  • 14-3-3Gamma interacts with and is phosphorylated by multiple protein kinase C isoforms in PDGF-stimulated human vascular smooth muscle cells.

    Autieri MV and Carbone CJ

    Department of Cardiology and Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. mautieri@unix.temple.edu

    It has recently been demonstrated that some members of the 14-3-3 protein family play an important role in signal transduction leading to cellular proliferation. We have previously shown that expression of 14-3-3gamma is induced by growth factors in human vascular smooth muscle cells (VSMC). In this study, we cloned the human homolog of 14-3-3gamma and observed many potential phosphorylation sites, suggesting the potential for post-translational modification. In VSMC treated with platelet-derived growth factor (PDGF), 14-3-3gamma protein was expressed and phosphorylated in an activation-dependent manner. Platelet-derived growth factor-induced phosphorylation could be inhibited by phosphokinase C (PKC) inhibitory compounds, and 14-3-3gamma could be phosphorylated in the absence of PDGF by compounds that activate PKC. We also demonstrated interaction between 14-3-3gamma and several PKC isoforms (alpha, beta, gamma, theta, and delta), implicating these PKC family isoforms as the kinases responsible for PDGF-induced 14-3-3gamma phosphorylation. We found that 14-3-3gamma interacted with the signal transduction protein Raf-1, suggesting that 14-3-3gamma provides a link between this protein and PKC. Thus, 14-3-3gamma may represent a signal transduction protein that is regulated transcriptionally and post-transcriptionally by growth factors.

    DNA and cell biology 1999;18;7;555-64

  • Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation.

    Fleming IN, Elliott CM, Buchanan FG, Downes CP and Exton JH

    Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.

    A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.

    The Journal of biological chemistry 1999;274;18;12753-8

  • Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion.

    Reed GL, Houng AK and Fitzgerald ML

    Cardiovascular Biology Laboratory, Harvard School of Public Health, Cardiac Unit, Massachusetts General Hospital, Boston, MA, USA. reed@cvlab.harvard.edu

    In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.

    Funded by: NHLBI NIH HHS: R01 HL57314-01

    Blood 1999;93;8;2617-26

  • Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals.

    Oancea E and Meyer T

    Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The specificity of many signal transduction pathways relies on the temporal coordination of different second messenger signals. Here we found a molecular mechanism which guarantees that conventional protein kinase C (PKC) isoforms are sequentially activated by calcium and diacylglycerol signals. Receptor stimuli that triggered repetitive calcium spikes induced a parallel repetitive translocation of GFP-tagged PKCgamma to the plasma membrane. While calcium acted rapidly, diacylglycerol binding to PKCgamma was initially prevented by a pseudosubstrate clamp, which kept the diacylglycerol-binding site inaccessible and delayed calcium- and diacylglycerol-mediated kinase activation. After termination of calcium signals, bound diacylglycerol prolonged kinase activity. The properties of this molecular decoding machine make PKCgamma responsive to persistent diacylglycerol increases combined with high- but not low-frequency calcium spikes.

    Funded by: NIGMS NIH HHS: GM-51457

    Cell 1998;95;3;307-18

  • Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis.

    Németh K, Salchert K, Putnoky P, Bhalerao R, Koncz-Kálmán Z, Stankovic-Stangeland B, Bakó L, Mathur J, Okrész L, Stabel S, Geigenberger P, Stitt M, Rédei GP, Schell J and Koncz C

    Abteilung Genetische Grundlagen der Pflanzenzüchtung, Federal Republic of Germany.

    The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-betaII isoenzyme involved in human insulin signaling.

    Genes & development 1998;12;19;3059-73

  • Elucidation of the exon-intron structure and size of the human protein kinase C beta gene (PRKCB).

    Greenham J, Adams M, Doggett N and Mole S

    Department of Paediatrics, University College London Medical School, The Rayne Institute, UK.

    As part of a transcriptional mapping project on human chromosome 16p12, a genomic contig was constructed that spanned the alternatively spliced human protein kinase C beta gene (PRKCB). PRKCB was determined to consist of 18 exons covering approximately 375 kb, with a particularly large intron of over 150 kb between exons 2 and 3. PRKCB is nearly 19 times larger than the highly homologous Drosophila melanogaster protein kinase C gene (dPKC), which has a similar-sized open reading frame but only 13 exons. This increase in size has occurred mostly as a result of expansion of introns, with intron size in the human gene averaging 22 kb compared with 1.5 kb in dPKC. The difference in gene size correlates with the difference in genome size, with the human haploid genome being nearly 18 times larger than the 170 Mb Drosophila haploid genome.

    Human genetics 1998;103;4;483-7

  • Protein kinase C phosphorylation of threonine at position 888 in Ca2+o-sensing receptor (CaR) inhibits coupling to Ca2+ store release.

    Bai M, Trivedi S, Lane CR, Yang Y, Quinn SJ and Brown EM

    Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+i dynamics in parathyroid cells.

    Funded by: NIDDK NIH HHS: DK09436, DK41415, DK48330; ...

    The Journal of biological chemistry 1998;273;33;21267-75

  • Effects of phosphorylation on function of the Rad GTPase.

    Moyers JS, Zhu J and Kahn CR

    Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, One Joslin Place, Boston, MA 02215, USA.

    Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases.

    Funded by: NIDDK NIH HHS: DK 45935, P30 DK36836, T32DK 07260

    The Biochemical journal 1998;333 ( Pt 3);609-14

  • Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways.

    Zidovetzki R, Wang JL, Chen P, Jeyaseelan R and Hofman F

    Department of Biology and Neuroscience, University of California, Riverside 92521, USA.

    The intracellular signal transduction pathways utilized by the HIV-1-derived protein, Tat, in the activation of human central nervous system-derived endothelial cells (CNS-ECs) were examined using specific enzymatic assays. Tat induced an increase in interleukin 6 (IL-6) mRNA within 1 hr of treatment. This biological effect of Tat involved activation of both protein kinase C (PK-C) and cAMP-dependent protein kinase (PK-A) in CNS-ECs. Tat at 10 ng/ml induced a sharp, transient increase in membrane PK-C activity within 30 sec of incubation, and reached maximum levels at 2 min, declining to control values within 10 min. Tat also induced a sharp increase in intracellular cAMP levels and PK-A activity in these cells, with the PK-A activity reaching a maximum at 10 min and slowly declining to control values in 4 hr of incubation. Activation of PK-A was dependent on a Tat-induced increase in membrane PK-C activity as demonstrated by calphostin C (a PK-C inhibitor) abolishing this effect. Incubation of cells with the cyclooxygenase inhibitor indomethacin did not affect Tat-induced activation of PK-A, indicating that prostacyclins are not involved in this process. Tat-induced increase in IL-6 mRNA was abolished in the presence on PK-A inhibitor H-89, demonstrating that activation of PK-A is necessary and sufficient for the increase in IL-6 production by these cells. Both the Tat-induced increase in intracellular cAMP and IL-6 mRNA levels in CNS-ECs may play a role in altering the blood-brain barrier and thereby inducing pathology often observed in AIDS dementia.

    Funded by: NINDS NIH HHS: NS33805

    AIDS research and human retroviruses 1998;14;10;825-33

  • Regulation of protein kinase C in the muscular layer of human placental stem villi vessels.

    Tertrin-Clary C, Fournier T and Ferré F

    INSERM U. 361, Université René Descartes, Paris, France. u361@cochin.inserm.fr

    Protein kinase C (PKC) activity in the muscular layer of stem villi vessels from the human term placenta was studied. Resting state PKC activity was distributed evenly between the cytosol and the particulate fractions. Upon stimulation by three different activators, phorbol 12-myristate 13-acetate, fluoride and endothelin-1, a translocation of PKC activity from the cytosolic to the particulate fraction was observed. The expression and distribution of PKC isoforms were then examined by Western blot analysis using specific antibodies to PKC isoforms. At least four PKC isoforms, PKCalpha, PKCbeta1, PKCbeta2, PKCzeta, and trace amounts of PKCepsilon were detected in both fractions. Their relative responses to the different agonists were examined by quantifying their subcellular redistribution. No significant differential activation of the four mainly expressed PKC isoforms were observed in response to stimulation with any of the stimuli. Moreover, our results show that endothelin-1 induced translocation/activation of PKC in this vascular smooth muscle.

    FEBS letters 1998;422;1;123-8

  • Extracellular HIV-1 Tat protein induces a rapid and selective activation of protein kinase C (PKC)-alpha, and -epsilon and -zeta isoforms in PC12 cells.

    Borgatti P, Zauli G, Cantley LC and Capitani S

    Division of Signal Transduction, Harvard Institute of Medicine, Beth Israel Hospital, Boston, Massachusettes 02115, USA.

    The addition in culture of extracellular HIV-1 Tat protein (0.1-1 nM) to PC12 cells induced a rapid increase of the bulk protein kinase C (PKC) catalytic activity. Among various PKC isoforms (alpha, beta I, beta II, delta, epsilon, eta, theta, and zeta) expressed in PC12 cells, Tat selectively stimulated alpha, epsilon, and zeta, as judged by activities in immunoprecipitates. Activation of these isoforms was suppressed by the tyrosine kinase inhibitor genistein. Moreover, PKC-zeta showed the fastest kinetics of activation in response to Tat, but PKC-alpha and PKC-epsilon showed the highest levels of activation. PKC-alpha activation was accompanied by a rise of intracellular IP3, while the PI 3-kinase inhibitors wortmannin and LY294002 suppressed PKC-epsilon activation. Taken together, these findings demonstrate that extracellular Tat shows a cytokine-like activity in PC12 cells, being able to trigger an intracellular signalling cascade which involves PKC-alpha, -epsilon, and -zeta.

    Biochemical and biophysical research communications 1998;242;2;332-7

  • Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain.

    Oh ES, Couchman JR and Woods A

    Department of Cell Biology, and Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, 35294, USA.

    Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus sequence. We investigated phosphorylation of syndecan-2 cytoplasmic domain by PKC, using purified GST-syndecan-2 fusion proteins and synthetic peptides corresponding to regions of the cytoplasmic domain. A synthetic peptide encompassing the entire cytoplasmic domain of syndecan-2 was phosphorylated by PKC with high affinity. Peptide mapping and substitution studies showed that both serines were phosphoacceptors, but each had slightly different affinity, with that of serine-197 being higher than serine-198. The efficiency of phosphorylation was concentration-dependent. At low concentrations, the cytoplasmic domain peptides were monomeric, with 2 mol/mol serine phosphorylation. At higher concentrations, however, the peptides formed dimers, with only 0.5 mol/mol phosphorylation. Concentration-dependent dimerization was not altered by phosphorylation. Phosphorylation is, therefore, dependent on the conformation of syndecan-2 cytoplasmic domain, but does not affect its oligomeric status.

    Funded by: NIAMS NIH HHS: P60 AR20614; NIGMS NIH HHS: GM50194

    Archives of biochemistry and biophysics 1997;344;1;67-74

  • Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level.

    Webb BL, Lindsay MA, Seybold J, Brand NJ, Yacoub MH, Haddad EB, Barnes PJ, Adcock IM and Giembycz MA

    Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London, UK.

    The protein kinase C (PKC) isoenzymes expressed by human peripheral lung and tracheal smooth muscle resected from individuals undergoing heart-lung transplantation were identified at the protein and mRNA level. Western immunoblot analyses of human lung identified multiple PKC isoenzymes that were differentially distributed between the soluble and particulate fraction. Thus, PKC alpha, PKC betaII, PKC epsilon, and PKC zeta were recovered predominantly in the soluble fraction whereas the eta isoform was membrane-associated together with trace amounts of PKC alpha and PKC epsilon. PKC beta1-like immunoreactivity was occasionally seen although the intensity of the band was uniformly weak. Immunoreactive bands corresponding to PKCs gamma, delta, or theta were never detected. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA extracted from human lung using oligonucleotide primer pairs that recognise unique sequences in each of the PKC genes amplified cDNA fragments that corresponded to the predicted sizes of PKC alpha, PKC betaI, PKC betaII, PKC epsilon, PKC zeta, and PKC eta (consistent with the expression of PKC isoenzyme protein) and, in addition, mRNA for PKC delta; PCR fragments of the expected size for the supposedly muscle-specific isoform, PKC theta, or the atypical isoenzyme, PKC lambda, were never obtained. The complement and distribution of PKC isoforms in human trachealis were similar, but not identical, to human lung. Thus, immunoreactive bands corresponding to the alpha, betaI, betaII, epsilon, and zeta isoenzymes of PKC were routinely labelled in the cytosolic fraction. In the particulate material PKC alpha, PKC epsilon, PKC alpha, PKC eta, and PKC mu were detected by immunoblotting. With the exception of PKC zeta, RT-PCR analyses confirmed the expression of the PKC isoforms detected at the protein level and, in addition, identified mRNA for PKC delta. Collectively, these data clearly demonstrate the expression of multiple PKC isoenzymes in human lung and tracheal smooth muscle, suggesting that they subserve diverse multifunctional roles in these tissues.

    Biochemical pharmacology 1997;54;1;199-205

  • Phosphorylation of vitronectin on Ser362 by protein kinase C attenuates its cleavage by plasmin.

    Gechtman Z and Shaltiel S

    Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel.

    Vitronectin, found in the extracellular matrix and in circulating blood, has an important role in the control of plasminogen activation. It was shown to be the major protein substrate in human blood fluid for a protein kinase A (PKA) released from platelets upon their physiological stimulation with thrombin. Since vitronectin was shown to have only one PKA phosphorylation site, but to contain 2-3 mol covalently bound phosphate, it was reasonable to assume that other protein kinases might phosphorylate vitronectin at other sites in the protein. We have reported earlier that human serum contains at least three protein kinases, one of which was found to be cAMP independent and to phosphorylate a repertoire of plasma proteins that was very similar to that obtained upon phosphorylation of human plasma with protein kinase C (PKC). Since there are now several examples of proteins with extracellular functions that are phosphorylated by PKC, we undertook to study the phosphorylation of vitronectin by PKC. Here, we show that vitronectin is a substrate for PKC, and characterize the kinetic parameters of this phosphorylation (Km approximately tenfold lower than the concentration of vitronectin in blood), indicating that, from the biochemical point of view, this phosphorylation can occur at the locus of a hemostatic event. We also identify Ser362 as the major PKC phosphorylation site in vitronectin, and confirm this localization by means of synthetic peptides derived from the cluster of basic amino acids in vitronectin surrounding Ser362. We show that the PKC phosphorylation at Ser362 alters the functional properties of vitronectin, attenuating its cleavage by plasmin at Arg361-Ser362. This phosphorylation has the potential to regulate plasmin production from plasminogen by a feedback mechanism involving the above-mentioned plasmin cleavage, a loosening of the vitronectin grip on inhibitor 1 of plasminogen activators, and a subsequent latency of this regulatory inhibitor.

    European journal of biochemistry 1997;243;1-2;493-501

  • Identification of a phosphorylation site for calcium/calmodulindependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.

    Omkumar RV, Kiely MJ, Rosenstein AJ, Min KT and Kennedy MB

    Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.

    The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L. -Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was approximately 50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons.

    Funded by: NIMH NIH HHS: MH49176; NINDS NIH HHS: NS17660, NS28710

    The Journal of biological chemistry 1996;271;49;31670-8

  • Protein-protein interaction of zinc finger LIM domains with protein kinase C.

    Kuroda S, Tokunaga C, Kiyohara Y, Higuchi O, Konishi H, Mizuno K, Gill GN and Kikkawa U

    Biosignal Research Center, Kobe University, Kobe 657, Japan. skuroda@inherit.biosig.kobe-u.ac.jp

    The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to associate with PKC in an isoform-specific manner. Deletion analysis demonstrated that any single LIM domain of ENH associates with the NH2-terminal region of PKC. ENH associated with PKC in COS-7 cells and was phosphorylated by PKC in vitro. Upon treatment of the cells with phorbol ester, ENH in the membrane fraction was translocated to the cytosol fraction in vivo. Other LIM domain-containing proteins, such as Enigma and LIM-kinase 1, also interacted with PKC through their LIM domains. These results suggest that the LIM domain is one of the targets of PKC and that the LIM-PKC interaction may shed light on undefined roles of LIM domain-containing proteins.

    The Journal of biological chemistry 1996;271;49;31029-32

  • In vitro phosphorylation of human immunodeficiency virus type 1 Tat protein by protein kinase C: evidence for the phosphorylation of amino acid residue serine-46.

    Holmes AM

    Biochemistry Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.

    Human immunodeficiency virus type-1 Tat protein is phosphorylated by protein kinase C in a calcium-, diacylglycerol-, and phosphatidylserine-dependent manner. Maximum phosphorylation is reached at a stoichiometry of between 0.45 and 0.5 mol of phosphate per mol of Tat. Several Tat peptides, containing serine at position 46, are the only ones which are phosphorylated at significant rates. Several other Tat peptides containing potential protein kinase C phosphorylation sites are not phosphorylated.

    Archives of biochemistry and biophysics 1996;335;1;8-12

  • Galpha12 and galpha13 are phosphorylated during platelet activation.

    Offermanns S, Hu YH and Simon MI

    Division of Biology 147-75, California Institute of Technology, Pasadena, California 91125, USA.

    The ubiquitously expressed G-proteins G12 and G13 whose function is currently not clear have been shown to be activated in platelet membranes through receptors that stimulate platelet aggregation. We used intact human platelets to determine whether alpha subunits of both G-proteins can be phosphorylated under physiological conditions. Activation of human platelets by thrombin and the thromboxane A2 receptor agonist U46619 lead to phosphorylation of Galpha12 and Galpha13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alphaIIbbeta3) activation. Phosphorylation of Galpha12 and Galpha13 could be mimicked by phorbol 12-myristate 13-acetate, and thrombin-induced phosphorylation was inhibited by the protein kinase C inhibitor calphostin C indicating an involvement of protein kinase C in Galpha12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with Galpha12 or Galpha13 and different protein kinase C isoforms. Among the protein knase C isoforms tested, protein kinase C beta, delta, and epsilon were most effective in promoting phosphorylation of Galpha12 and Galpha13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Galpha12 and Galpha13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.

    The Journal of biological chemistry 1996;271;42;26044-8

  • Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexin II.

    Jost M and Gerke V

    University of Münster, Clinical Research Group for Endothelial Cell Biology, Münster, Germany.

    Annexin II is a Ca(2+)-regulated membrane- and cytoskeleton-binding protein implicated in membrane transport events along the Ca(2+)-regulated secretory and the early endocytic pathway. Biochemical properties of this annexin and its intracellular distribution are regulated by complex formation with p11 (S100A10), a member of the S100 protein family. The annexin II-p11 interaction is mediated through the unique N-terminal domain of annexin II and is inhibited by protein kinase C phosphorylation of a serine residue in annexin II. To map this regulatory serine phosphorylation site we developed a baculovirus-mediated expression system for wild-type annexin II and for a series of annexin II mutants which contained substitutions in one or more serine residues present in the N-terminal domain. The different mutant derivatives were purified and shown to display the same biochemical properties as recombinant wild-type annexin II and the authentic protein purified from porcine intestine. However, significant differences in phosphate incorporation were observed when the individual serine mutants were subjected to phosphorylation by protein kinase C. A comparison of the phosphorylation patterns obtained identified Ser-II as the protein kinase C site responsible for regulating the annexin II-p11 interaction. Ser-II lies within the sequence mediating p11 binding, i.e. amino-acid residues 1 to 14 of annexin II, and phosphorylation at this site most likely leads to a direct spatial interference with p11 binding.

    Biochimica et biophysica acta 1996;1313;3;283-9

  • Differential expression of protein kinase C isoforms in the human placenta.

    Ruzycky AL, Jansson T and Illsley NP

    Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, USA.

    The extensive role played by protein kinase C (PKC) in signal transduction prompted this study of the expression and localization of PKC isoforms in human placental syncytiotrophoblast. Membranes prepared from these cells and samples of villous tissue were analysed by immunoblotting and immunocytochemistry using isoform-specific antibodies. PKC beta 2, gamma, epsilon and zeta were found to be present in both microvillous and basal membranes from term placenta. The alpha isoform was observed only on the basal membrane while the beta 1 isoform was confined to the microvillous membrane. The basal microvillous ratios for beta 2, gamma, epsilon and zeta ranged between 0.3 and 0.5, demonstrating a substantial asymmetry in plasma membrane localization. Immunocytochemistry supported the isoform identification and localization observed in the immunoblotting experiments. Moreover the cellular distribution showed that the majority of syncytical PKC was bound to the plasma membranes, in contrast to the other villous cell types. Immunoblotting experiments demonstrated significant increases in PKC beta 2 and epsilon on the microvillous membrane and PKC gamma and epsilon on the basal membrane between 16 and 40 of weeks gestation. This is the first detailed mapping of PKC isoform distribution in an epithelial cell type and demonstrates the potential for selectivity in signal transduction through phosphorylation of isoform specific and spatially-separated substrates.

    Funded by: NICHD NIH HHS: R01 HD23498

    Placenta 1996;17;7;461-9

  • Phosphorylation of GAP-43 (growth-associated protein of 43 kDa) by conventional, novel and atypical isotypes of the protein kinase C gene family: differences between oligopeptide and polypeptide phosphorylation.

    Oehrlein SA, Parker PJ and Herget T

    Institute of Physiological Chemistry, Johannes-Gutenberg University, Mainz, Germany.

    GAP-43 (growth-associated protein of 43 kDa; also known as neuromodulin, P-57, B-50 and F-1) is a neuronal calmodulin binding protein and a major protein kinase C (PKC) substrate in mammalian brain. Here we describe the phosphorylation by and the site specificity of different PKC isotypes. The conventional PKC beta 1 and the novel PKCs delta and epsilon effectively phosphorylated recombinant GAP-43 in vitro; atypical PKC zeta did not. The K(m) values (between 0.6 and 2.3 microM) were very low, demonstrating a high-affinity interaction between kinase and substrate. All PKC isotypes were shown to phosphorylate serine-41 in GAP-43. When using a 19-amino-acid oligopeptide based on the GAP-43 phosphorylation site as substrate, there was a significant difference compared with polypeptide phosphorylation. The V(max) values of PKC beta 1 and PKC epsilon were much higher for this oligopeptide than for the complete protein (up to 10-fold); in contrast, their apparent affinities for the peptide were much lower (up to 100-fold) than for the intact GAP-43 polypeptide. Furthermore, phosphorylation of the GAP-43 oligopeptide by PKC beta 1 was more sensitive to a catalytic-site inhibitor than was phosphorylation of intact GAP-43. These results suggest that there are multiple sites of interaction between GAP-43 and PKC.

    The Biochemical journal 1996;317 ( Pt 1);219-24

  • Protein kinase C tissue localization in human colonic tumors suggests a role for adenoma growth control.

    Kahl-Rainer P, Sedivy R and Marian B

    Institute of Tumor Biology-Cancer Research, University of Vienna Medical School, Austria.

    Protein kinase C (PKC) has been implicated as a mediator of growth control during colorectal carcinogenesis, but the mechanisms involved are still a matter of dispute. The aim of this study was to analyze PKC patterns and tissue distribution to gain further insight in PKC function during tumor development in the gut.

    Methods: PKC isoenzymes alpha, beta 1, beta 2, delta, and eta and the proliferation antigen Ki67 were analyzed in formalin-fixed normal, premalignant, and malignant specimens using immunohistochemistry.

    Results: In normal colonic mucosa, protein levels of all PKC isoenzymes followed an increasing gradient from the bottom to the top of the crypt, staining mainly terminally differentiated, resting cells. Atypical crypts observed in the normal mucosa adjacent to tumors expressed higher levels of Ca(2+)-dependent isoenzymes than the surrounding tissue. In tumors, the number and abundance of PKC isoenzymes was inversely related to proliferation in 7 adenomas and 9 carcinomas. Areas containing PKC-beta 1 as the only isoenzyme had the highest proliferation rates (50%-82% Ki67-positive cells).

    Conclusions: The data suggest a function of PKCs, especially PKC-beta 1, in colorectal carcinogenesis and tumor growth control.

    Gastroenterology 1996;110;6;1753-9

  • Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4E-binding proteins.

    Whalen SG, Gingras AC, Amankwa L, Mader S, Branton PE, Aebersold R and Sonenberg N

    Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.

    Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure (m7GpppX, where X is any nucleotide) that binds the multisubunit initiation factor eIF4F through one of its subunits, eIF4E. eIF4E is a phosphoprotein whose phosphorylation state positively correlates with cell growth. Protein kinase C phosphorylates eIF4E in vitro, and possibly in vivo. Using recombinant eIF4E incubated in vitro with purified protein kinase C and analyzed by solid-phase phosphopeptide sequencing in combination with high performance liquid chromatography coupled to mass spectrometry, we demonstrated that the third amino acid of the peptide SGSTTK (Ser209) is the major site of phosphorylation. This finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E (Joshi, B., Cai, A. L., Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J., Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E. (1995) J. Biol. Chem. 270, 14597-14603). A S209A mutation resulted in dramatically reduced phosphorylation, both in vitro and in vivo. Furthermore, the mutant protein was phosphorylated on threonine (most probably threonine 210) in vivo. Here we show that in the presence of the recently characterized translational repressors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase C is strongly reduced. This suggests a two-step model for the phosphorylation (and activation) of eIF4E by growth factors and hormones: first, dissociation of eIF4E from 4E-BPs, followed by eIF4E phosphorylation.

    The Journal of biological chemistry 1996;271;20;11831-7

  • Increased expression of protein kinase C beta activates ERK3.

    Sauma S and Friedman E

    Laboratory of Gastrointestinal Tumor Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

    In a prior study, we have shown that stable transfection of expression plasmids for protein kinases C beta 1 (PKC beta 1) or PKC beta 2 into differentiated colon cancer cells led to elevated levels of PKC beta 1 or PKC beta 2 protein and PKC beta kinase activities in the transfectants, without altering PKC alpha levels. PKC gamma is not found in these cells, so the major modulation was in PKC beta. PKC beta transfectant cells exhibited blocked differentiation, increased growth rate in athymic mice, and restoration of the basic fibroblast growth factor response pathway. In this study, we have extended the analysis of these PKC beta transfectants to the mitogen-activated protein kinase ERK3. Analysis of cell lysates on the mitogen-activated protein kinase substrate myelin basic protein by in gel kinase assay showed increased activity at 63 kDa, the size of ERK3, in each of two PKC beta 1 and each of two PKC beta 2 transfectants compared with the vector control transfectant. ERK3 was expressed at equal abundance in PKC beta 1, PKC beta 2, and control transfectant cells as demonstrated by Western blotting and by immunoprecipitation with anti-ERK3 monoclonal antibody. However, a > 10-fold increase in ERK3 activity in each PKC beta transfectant was shown by immunoprecipitation with anti-ERK3 monoclonal antibody followed by either immune complex kinase assay or by in gel kinase assay. Thus, while overexpression of transfected PKC beta does not lead to overexpression of ERK3, it does lead to constitutive activation of ERK3. A causal link between PKC beta overexpression and ERK3 activation was established because 12-O-tetradecanoylphorbol-13-acetate treatment down-regulated both PKC and ERK3 activities in both PKC beta 1 transfectants. ERK3 activity was found in nuclear and membrane fractions in each PKC beta transfectant, in contrast to controls, perhaps accounting for constitutive activation of ERK3 in cells with elevated levels of PKC beta 1 or PKC beta 2.

    Funded by: NCI NIH HHS: R01 CA45783, R01 CA67405

    The Journal of biological chemistry 1996;271;19;11422-6

  • Persistent membrane translocation of protein kinase C alpha during 12-0-tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells.

    Powell CT, Brittis NJ, Stec D, Hug H, Heston WD and Fair WR

    George M. O'Brien Urology Research Center for Prostate Cancer, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

    Others have reported that the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator and down-regulator of most protein kinase C (PKC) isozymes, can induce apoptotic cell death of androgen-sensitive LNCaP but not androgen-insensitive PC-3 or DU 145 human prostate cancer cells. As a first step toward uncovering the mechanism by which TPA induces apoptosis of LNCaP cells, we quantified expression of PKC isozyme mRNAs in unmodified and TPA-resistant LNCaP cells and in naturally TPA-resistant PC-3, PC-3M, and DU 145 cells. All of the cell lines and normal prostate expressed RNAs for PKC alpha, delta, epsilon, eta, and mu; only DU 145 cells and normal prostate expressed PKC beta and theta RNAs, and none expressed PKC gamma. The amount of PKC alpha RNA and protein was 6- to 38-fold lower, and PKC mu RNA was 4.5- to 16.5-fold higher in unmodified and TPA-resistant LNCaP cells than in the androgen-independent cells. We examined the effects of TPA on PKC alpha and mu mRNA levels and on membrane translocation of PKC alpha. Incubation with TPA for 6 h or more induced 95% inhibition of cell growth, a transient 12-fold increase and 5-fold decrease in PKC alpha and mu mRNA levels, respectively, and prolonged translocation of PKC alpha to non-nuclear membranes in unmodified LNCaP cells and in TPA-resistant LNCaP cells from which TPA had been removed for 10 days. TPA-resistant LNCaP cells in the continuous presence of TPA, or 24 h after removal of TPA, had down-regulated PKC alpha and remained resistant to re-addition of TPA. These data demonstrate a strong correlation of the presence and absence of membrane PKC alpha with apoptosis and resistance to apoptosis, respectively.

    Funded by: NIDDK NIH HHS: DK/CA47650

    Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 1996;7;4;419-28

  • Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C: its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with syntaxin.

    Fujita Y, Sasaki T, Fukui K, Kotani H, Kimura T, Hata Y, Südhof TC, Scheller RH and Takai Y

    Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Japan.

    Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport. On the other hand, protein kinase C (PKC) stimulates Ca2+-dependent exocytosis in various types of secretory cells. However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified. Here, we show that recombinant Munc-18 is phosphorylated by conventional PKC in a Ca2+- and phospholipid-dependent manner in a cell-free system. About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. The major phosphorylation sites are Ser306 and Ser313. The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc-18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane.

    The Journal of biological chemistry 1996;271;13;7265-8

  • Extracellular human immunodeficiency virus type 1 Tat protein is associated with an increase in both NF-kappa B binding and protein kinase C activity in primary human astrocytes.

    Conant K, Ma M, Nath A and Major EO

    Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.

    Human immunodeficiency virus type 1 (HIV-1) infection has been associated with an increase in the binding of the transcription factor NF-kappa B to its consensus sequence in the viral promoter. Using cultures of primary human fetal astrocytes, we show that exogenous HIV-1 Tat protein, which has been demonstrated to be released from infected cells, is associated with an increase in the binding of this transcription factor to an HIV-1 long terminal repeat kappa B sequence. This effect occurs rapidly and is independent of new protein synthesis. We also demonstrate that extracellular Tat protein is associated with an increase in protein kinase C activity. If Tat functions similarly in other cell types, such findings could relate to some of this protein's previously described physiological effects. These effects include Tat's ability to upregulate the synthesis of specific cytokines and to act as a growth factor.

    Journal of virology 1996;70;3;1384-9

  • IL-16- and other CD4 ligand-induced migration is dependent upon protein kinase C.

    Parada NA, Cruikshank WW, Danis HL, Ryan TC and Center DM

    Department of Medicine, Pulmonary Center, Boston University School of Medicine, MA 02118, USA.

    Human interleukin-16, previously known as lymphocyte chemoattractant factor, is a CD4+ T cell competence growth factor initially described as a chemotactic factor for CD4+ cells. The interaction between IL-16 and its receptor CD4 leads to an increase in intracytoplasmic calcium and inositol triphosphate. Because of the association of intracellular shifts in protein kinase C (PKC) enzyme activity with production of these secondary messengers and the participation of PKC in transducing certain receptor-mediated migratory signals, we investigated the role of PKC in the CD4-mediated migratory response by IL-16. Recombinant IL-16 induces rapid translocation of PKC from the cytosol to the membrane in three separate CD4+ cell types: normal blood T cells, SUPT1 cells, and THP1 cells. PKC inhibitors H7, calphostin C, chelerythrine, and bisindolylmaleimide completely block IL-16-induced lymphocyte migration as well as the motile response induced by HIV-1 gp120 and anti-CD4 antibodies. Taken together, these data suggest a role for PKC in CD4-mediated migratory responses.

    Funded by: NHLBI NIH HHS: P50 HL46563, R01 HL32802

    Cellular immunology 1996;168;1;100-6

  • PRK1 phosphorylates MARCKS at the PKC sites: serine 152, serine 156 and serine 163.

    Palmer RH, Schönwasser DC, Rahman D, Pappin DJ, Herget T and Parker PJ

    Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, London, UK.

    The 80kDa Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is a major in vivo substrate of protein kinase C (PKC). Here we report that MARCKS is a major substrate for the lipid-activated PKC-related kinase (PRK1) in cell extracts. Furthermore, PRK1 is shown to phosphorylate MARCKS on the same sites as PKC in vitro. Thus, control of MARCKS phosphorylation on these previously identified 'PKC' sites may be regulated under certain circumstances by PRK as well as PKC mediated signalling pathways. The implications for MARCKS as a marker of PKC activation and as a point of signal convergence are discussed.

    FEBS letters 1996;378;3;281-5

  • Activation of human platelet protein kinase C-beta 2 in vivo in response to acute hyperglycemia.

    Pirags V, Assert R, Haupt K, Schatz H and Pfeiffer A

    Medizinische Universitätsklinik Bergmannsheil, Ruhr-University, Bochum, Germany.

    Protein kinase C (PKC) is known to be activated in experimental model systems by elevated glucose and may play an important role in the pathogenesis of diabetic complications. Since there is no information about its role in humans in vivo we investigated the activation of PKC in human thrombocytes during infusion of glucose and insulin in normal controls and in 19 NIDDM patients by determining membrane and cytosol levels of PKC beta 2 using immune blots. In the 27 subjects investigated (8 controls, 19 NIDDM) membrane-associated levels of PKC beta 2 increased significantly after 60 and 150 min (p < 0.005). In controls an increase of membrane and of cytosolic PKC beta 2 occurred upon elevation of glucose by 5.5 mmol/L or more and the membrane association persisted for at least 60 min. In NIDDM glucose was elevated by 7.5-10 mmol/L during infusions. Increases of both membrane and cytosolic PKC beta 2 (< 20%-300%) occurred in 10 NIDDM patients suggesting that both, translocation and increased synthesis of PKC beta 2 were stimulated by glucose. Nine other patients showed no alteration (i.e. < 20%) of PKC beta 2. The 2 groups were similar regarding parameters of diabetes control, baseline glucose and glucose elevation during the test. However, the PKC beta 2 responsive group had lower levels of serum triglycerides (1.39 +/- 0.19 vs. 2.32 +/- 0.34 g/L; p = 0.038). To assess whether absolute levels of PKC were altered in human diabetes, platelet levels of PKC alpha, beta 1 and beta 2 were determined in 22 controls and 25 NIDDM subjects with poorly controlled diabetes (HbA1c = 9.8 +/- 0.36%). Cytosolic levels of PKC alpha were significantly decreased by 27% compared to controls in NIDDM but there was no change of PKC beta 1 or PKC beta 2. We conclude that 1. acute elevation of glucose by 5.5 mmol/L or more can activate PKC beta 2 translocation in controls and NIDDM patients in vivo irrespective of parameters of metabolic control. 2. NIDDM patients differ in their PKC beta 2-responses to glucose and 3. poor metabolic control leads to moderate downregulation of PKC alpha suggesting continued activation.

    Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 1996;104;6;431-40

  • Protein kinase C is regulated in vivo by three functionally distinct phosphorylations.

    Keranen LM, Dutil EM and Newton AC

    Department of Pharmacology, University of California at San Diego, La Jolla 92093-0640, USA.

    Background: Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers.

    Results: We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C beta II are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site.

    Conclusions: Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.

    Funded by: NIGMS NIH HHS: GM 43154

    Current biology : CB 1995;5;12;1394-1403

  • Cell cycle phase-specific phosphorylation of human topoisomerase II alpha. Evidence of a role for protein kinase C.

    Wells NJ, Fry AM, Guano F, Norbury C and Hickson ID

    Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, United Kingdom.

    Type II topoisomerases are essential for faithful cell division in all organisms. In human cells, the alpha isozyme of topoisomerase II has been implicated in catalyzing mitotic chromosome segregation via its action as a DNA unlinking enzyme. Here, we have shown that the enzymatic activity of topoisomerase II alpha protein purified from HeLa cell nuclei was strongly enhanced following phosphorylation by protein kinase C. We have investigated the possibility that this kinase is involved in cell cycle phase-specific phosphorylation of topoisomerase II alpha in HeLa cells. Two-dimensional tryptic phosphopeptide mapping revealed that topoisomerase II alpha protein immunoprecipitated from metabolically labeled HeLa cells was differentially phosphorylated during the G2/M phases of the cell cycle. To identify sites of phosphorylation, and the kinase(s) responsible for this modification, oligohistidine-tagged recombinant domains of topoisomerase II alpha protein were overexpressed in Escherichia coli and purified by affinity chromatography. Phosphorylation of a short fragment of the N-terminal ATPase domain of topoisomerase II alpha by protein kinase C in vitro generated two phosphopeptides that co-migrated with prominent G2/M phase-specific phosphopeptides from the HeLa cell-derived topoisomerase II alpha protein. Site-directed mutagenesis studies indicated that phosphorylation of serine 29 generated both of these phosphopeptides. Our results implicate protein kinase C in the cell cycle phase-dependent modulation of topoisomerase II alpha enzymatic activity in human cells.

    The Journal of biological chemistry 1995;270;47;28357-63

  • The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C.

    Herget T, Oehrlein SA, Pappin DJ, Rozengurt E and Parker PJ

    Institute of Physiological Chemistry, University of Mainz, Germany.

    The myristoylated alanine-rich C-kinase substrate (MARCKS) is the major protein kinase C (PKC) substrate in many cell types including fibroblasts and brain cells. Here we describe the phosphorylation of MARCKS and the site specificity for different PKC isotypes. Conventional (c)PKC beta 1, novel (n)PKC delta and nPKC epsilon efficiently phosphorylated the MARCKS protein in vitro. The Km values were extremely low, reflecting a high affinity between kinases and substrate. The apparent affinity of nPKC delta (Km = 0.06 microM) was higher than that of nPKC epsilon and cPKC beta 1 (Km = 0.32 microM). The rate of substrate phosphorylation was inversely correlated with affinity and decreased in the order nPKC epsilon > cPKC beta 1 > nPKC delta. Atypical (a)PKC zeta did not phosphorylate the intact MARCKS protein. However, a 25-amino-acid peptide deduced from the MARCKS phosphorylation domain, was efficiently phosphorylated by aPKC zeta as well as by the other three PKC. Site analysis revealed that only serine residues S152, S156 and S163 were phosphorylated, with S163 phosphorylated highest, followed by S156 and S152; in contrast, S160 and S167 were not phosphorylated. No further PKC phosphorylation sites could be detected in MARCKS. The phosphorylation pattern was independent of the type of PKC isotype used. Kinetic analysis showed, that MARCKS is sequentially phosphorylated in the order S156 > S163 > S152 by cPKC, nPKC and aPKC. There was no dramatic difference in the sequential phosphorylation of MARCKS detectable when comparing the four PKC isotypes. The results are discussed in the context of the functional significance of MARCKS phosphorylation.

    European journal of biochemistry 1995;233;2;448-57

  • HIV-1 envelope glycoproteins induce activation of activated protein-1 in CD4+ T cells.

    Chirmule N, Goonewardena H, Pahwa S, Pasieka R, Kalyanaraman VS and Pahwa S

    Department of Pediatrics, North Shore University Hospital, Cornell University Medical College, Manhasset, New York 11030, USA.

    Activation of CD4 positive T cells is a primary requirement for human immunodeficiency virus (HIV) entry, efficient HIV replication, and progression to AIDS, Utilizing CD4 positive T cell lines and purified T cells from normal individuals, we have demonstrated that native envelope glycoproteins of HIV, gp 160, can induce activation of transcription factor, activated protein-1 (AP-1). The stimulatory effects of gp160 are mediated through the CD4 molecule, since treatment of gp160 with soluble CD4-IgG abrogates its activity, and CD4 negative T cell lines fail to be stimulated with gp160. Immunoprecipitation of the gp 160-induced nuclear extracts with polyclonal antibodies to Fos and Jun proteins indicates that AP-1 complex is comprised of members of these family of proteins. The gp160-induced AP-1 complex is dependent upon protein tyrosine phosphorylation and is protein synthesis-independent. This stimulation can also be abolished by inhibitors of protein kinase C, but it is unaffected by calcium channel blocker or cyclosporine A. This gp160 treatment adversely affects the functional capabilities of T cells: pre-treatment of CD4+ T cells with gp160 for 4 h at 37 degrees C inhibited anti-CD3-induced interleukin-2 secretion. Effects similar to gp160 were seen with anti-CD4 mAb. The aberrant activation of AP-1 by gp160 in CD4 positive T cells could result in up-regulation of cytokines containing AP-1 sites, e.g. interleukin-3 and granulocyte macrophage colony-stimulating factor, and concurrently lead to T cell unresponsiveness by inhibiting interleukin-2 secretion.

    Funded by: NCRR NIH HHS: MO1 RR 0047; NIAID NIH HHS: AI 28281, AI 35414

    The Journal of biological chemistry 1995;270;33;19364-9

  • Localization of protein kinases by anchoring proteins: a theme in signal transduction.

    Mochly-Rosen D

    Department of Molecular Pharmacology, School of Medicine, Stanford University, CA 94305-5332, USA.

    A fundamental question in signal transduction is how stimulation of a specific protein kinase leads to phosphorylation of particular protein substrates throughout the cell. Recent studies indicate that specific anchoring proteins located at various sites in the cell compartmentalize the kinases to their sites of action. Inhibitors of the interactions between kinases and their anchoring proteins inhibit the functions mediated by the kinases. These data indicate that the location of these anchoring proteins provides some of the specificity of the responses mediated by each kinase and suggest that inhibitors of the interaction between the kinases and their anchoring proteins may be useful as therapeutic agents.

    Funded by: NHLBI NIH HHS: R01 HL-43380

    Science (New York, N.Y.) 1995;268;5208;247-51

  • Characterization of Rad, a new member of Ras/GTPase superfamily, and its regulation by a unique GTPase-activating protein (GAP)-like activity.

    Zhu J, Reynet C, Caldwell JS and Kahn CR

    Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215.

    We have recently identified a new member of the Ras/GTPase superfamily termed Rad which has unique sequence features and is overexpressed in the skeletal muscle of humans with type II diabetes (Reynet, C., and Kahn, C. R. (1993) Science, 262, 1441-1444). When expressed in bacteria as a glutathione S-transferase fusion protein, Rad bound [alpha-32P]GTP quickly and saturably. Binding was specific for guanine nucleotides and displayed unique magnesium dependence such that both GTP and GDP binding were optimal at relatively high Mg2+ concentrations (1-10 mM). Rad had low intrinsic GTPase activity which was greatly enhanced by a GTPase-activating protein (GAP) activity present in various tissues and cell lines. Several known GAPs had no stimulatory effect toward Rad. Conversion of Ser to Asn at position 66 in Rad (equivalent to position 12 in Ras) resulted in a total loss of GTP binding. Mutation of Pro61 (equivalent to Gly12 in Ras) or Gln109 (equivalent to Gln61 in Ras) had no effect on Rad GTPase activity, whereas creation of a double mutation at these positions resulted in exceptionally high intrinsic GTPase activity. In vitro, Rad was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PK). Phosphopeptide mapping indicated two PKA phosphorylation sites near the COOH terminus. Rad also co-precipitated a serine/threonine kinase activity from extracts of various tissues and cell lines which catalyzed phosphorylation on Rad but was not inhibited by PKA inhibitor. Thus, Rad is a GTP-binding protein and a GTPase which has some structure/function similarities to Ras, but displays unique features. Rad may also be phosphorylated on serine/threonine residues by PKA and other kinases, as well as regulated by its own GAP which is present in many tissues and cell types.

    Funded by: NIDDK NIH HHS: DK 36836, DK 45935

    The Journal of biological chemistry 1995;270;9;4805-12

  • Autoregulation of cloned human protein kinase C beta and gamma gene promoters in U937 cells.

    Mahajna J, King P, Parker P and Haley J

    Ludwig Institute for Cancer Research, London, UK.

    Protein kinase C (PKC) serine/threonine kinases transduce cellular signals initiated by phospholipase C activation and diacylglycerol production. Human gene sequences from the beta and gamma isoforms were cloned and sequenced, and transcriptional regulation was studied. The major PKC beta transcription initiation site was identified by primer extension and S1 nuclease protection. Additional transcription initiation sites were located within a CpG-rich region proximal to the ATG. The transcription initiation site of the PKC gamma gene was identified by primed cDNA synthesis. In transfection experiments, the PKC gamma promoter was expressed at high level in U937 and HL60 cells but not in COS-1 cells. A sequence motif (AnAGATTCanAGAGnCa), reiterated over at least 1 kb, was located approximately 1.5 kb 5' of the PKC gamma translation initiation codon. This repetitive motif is abundant in run-on RNA in the hematopoietic and epithelial cell lines tested. Analysis of promoter deletion constructs by transient transcription assays in U937, IM9, and COS-1 cells showed negative regulation of the PKC beta promoter by sequences located between -3,000 and -690. although no homology between PKC beta and PKC-gamma 5'-flanking sequences was observed, both PKC beta and PKC gamma promoters were potently induced by 12-phorbol 13-myristate in transfected U937 cells.

    DNA and cell biology 1995;14;3;213-22

  • Inhibition of protein kinase C by a synthetic peptide corresponding to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 envelope glycoprotein.

    Ward NE, Gravitt KR and O'Brian CA

    Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.

    This report describes the inhibition of protein kinase C (PKC) by a synthetic peptide corresponding to a viral sequence expressed in mammalian cells. The peptide corresponds to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp41), and it inhibits Ca(2+)- and phosphatidylserine (PS)-dependent phosphorylation of synthetic peptide substrates and histone by purified PKC with IC50 values ranging from 9 to 32 microM. Although previously described pKC-inhibitory synthetic peptides corresponding to sequences expressed in mammalian cells are also effective against the phosphorylation of synthetic peptide substrates, they fail to affect PKC-catalysed phosphorylation of potent protein substrates such as histone. This may limit their usefulness as inhibitors of PKC-catalysed protein phosphorylation in cellular systems. PKC activation is a major contributing factor in multidrug resistance (MDR) in cancer. Our observation that the synthetic peptide gp41(828-848) inhibits pKC-catalysed phosphorylation of a protein substrate suggests the potential value of expressing the viral sequence gp41(828-848) in cancer cells as a novel in vitro model system of MDR reversal.

    Funded by: NCI NIH HHS: CA-52460

    Cancer letters 1995;88;1;37-40

  • Requirement for negative charge on "activation loop" of protein kinase C.

    Orr JW and Newton AC

    Department of Chemistry, Indiana University, Bloomington 47405.

    Increasing evidence has implicated a post-translational phosphorylation in the production of a catalytically competent protein kinase C. Here we present structural and biochemical evidence that Thr500 of protein kinase C-beta II is the residue phosphorylated by another kinase. Modeling studies indicate that this residue is part of a "lip" structure at the entrance of the catalytic site; phosphorylation on this lip, or "activation loop," is central to the regulation of three kinases whose structures have been elucidated (Taylor, S. S., and Radzio-Andzelm, E. (1994) Structure 2, 345-355). Biochemical data reveal that mutation of Thr500 to an acidic residue (Glu) results in expression of catalytically active protein kinase C in COS cells. In contrast, mutation of this residue to a neutral, non-phosphorylatable residue (Val) results in expression of inactive enzyme. Thus, negative charge at position 500 is required for catalytically competent protein kinase C-beta II. These data suggest that signal processing by protein kinase C cannot occur until the enzyme is first phosphorylated by a protein kinase C kinase.

    Funded by: NIGMS NIH HHS: GM 43154

    The Journal of biological chemistry 1994;269;44;27715-8

  • The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases.

    el Benna J, Faust LP and Babior BM

    Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.

    The respiratory burst oxidase catalyzes the production of O2.- from oxygen and NADPH. It is dormant in resting cells but becomes active when the cells are stimulated. Activation is accompanied by the phosphorylation of multiple serines in the cytosolic oxidase component p47phox, which moves from cytosol to the membrane during oxidase activation. Using immunopurified p47phox isolated from 32Pi-loaded neutrophils activated with phorbol myristate acetate, we showed that all the 32P was in the C-terminal CNBr fragment of the protein, and that in that fragment, Ser-303, Ser-304, Ser-320, Ser-328, Ser-345, and Ser-348 and at least one of the three serines, Ser-359, Ser-370, and Ser-379, were phosphorylated, while Ser-282, Ser-287, Ser-381, and Ser-388 were not. Of the phosphorylated serines, Ser-303, Ser-304, Ser-320, and Ser-328 are located in protein kinase C substrate sequences. Ser-345 and Ser-348, however, are located in sequences recognized by mitogen-activated protein (MAP) kinase (-PXSP-). This finding suggests that MAP kinase or a related proline-directed kinase may participate in the regulation of O2.- production by activated neutrophils. The tryptic peptide map of p47phox phosphopeptides from neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine closely resembled that of p47phox phosphopeptides from phorbol-activated cells, suggesting that the same serines were phosphorylated in response to each agent.

    Funded by: NIAID NIH HHS: AI-24227, AI-28479, AI-30742; ...

    The Journal of biological chemistry 1994;269;38;23431-6

  • Phosphorylation of Thr642 is an early event in the processing of newly synthesized protein kinase C beta 1 and is essential for its activation.

    Zhang J, Wang L, Schwartz J, Bond RW and Bishop WR

    Molecular Pharmacology Section, Schering-Plough Research Institute, Kenilworth, New Jersey 07003.

    Earlier studies of a site-specific mutant of protein kinase C beta 1 (PKC beta 1) altered at Thr635 and Thr642 indicated that these phosphorylation sites are critical for enzymatic function (Zhang, J., Wang, L., Petrin, J., Bishop, W. R., and Bond, R. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,6130-6134). To determine the contribution of the individual threonines, we report here on two site-specific mutants in which either Thr635 or Thr642 was changed to alanine. When transiently overexpressed in Cos cells wild-type PKC beta 1 exists in two forms: a Triton-insoluble form with high electrophoretic mobility and a slower migrating Triton-soluble form. Mutation at Thr642 (but not Thr635) results in production of only the fast-migrating form. [35S]Methionine pulse-chase labeling indicates that wild-type PKC beta 1 is synthesized as the fast-migrating form and is subsequently converted to the slow-migrating form. 32P labeling shows that only the slow-migrating form is a phosphoprotein. Mutation of Thr642 abolishes this phosphorylation. Finally, the Thr642 mutant PKC beta 1 lacks enzymatic activity and, when expressed in NIH 3T3 cells, reduces phorbol ester-induced c-fos promoter activity. These results indicate that Thr642 phosphorylation is an early event in the processing of newly synthesized PKC beta 1 and is required for enzymatic function. These results support a role for a PKC kinase in PKC processing and activation.

    The Journal of biological chemistry 1994;269;30;19578-84

  • Identification of nuclear beta II protein kinase C as a mitotic lamin kinase.

    Goss VL, Hocevar BA, Thompson LJ, Stratton CA, Burns DJ and Fields AP

    Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.

    Multisite phosphorylation of the nuclear lamins is thought to regulate the process of mitotic nuclear envelope breakdown in vivo. Here we investigate the involvement of two proposed human mitotic lamin kinases, beta II protein kinase C (PKC) and p34cdc2/cyclin B kinase, in human lamin B1 phosphorylation in vitro and in intact cells. We find that both kinases can phosphorylate purified soluble lamin B at similar rates. However, beta II PKC phosphorylates interphase nuclear envelope lamin B at more than 200 times the rate of human p34cdc2/cyclin B kinase. beta II PKC-mediated phosphorylation of lamin B is confined to two sites, Ser395 and Ser405, within the carboxyl-terminal domain, whereas human p34cdc2/cyclin B kinase phosphorylates a single site, Ser23, in the amino-terminal domain. A second potential p34cdc2/cyclin B kinase site within the carboxyl-terminal domain, Ser393, is not phosphorylated by human p34cdc2/cyclin B kinase. However, invertebrate p34cdc2/cyclin B kinase from sea star exhibits a different specificity, phosphorylating both amino- and carboxyl-terminal sites. Mitotic human lamin B from intact cells is phosphorylated predominantly in its carboxyl-terminal domain. Comparative tryptic phosphopeptide mapping demonstrates that the beta II PKC site, Ser405, is a prominent target of mitotic lamin B phosphorylation in vivo. beta II PKC translocates to the nucleus during the G2/M phase of cell cycle concomitant with phosphorylation of Ser405, indicating a physiologic role for nuclear beta II PKC activation in mitotic lamin B phosphorylation in vivo. The presence of phosphorylation sites within the carboxyl-terminal domain of mitotic lamin B which are not phosphorylated by either beta II PKC or p34cdc2/cyclin B kinase suggests the involvement of other lamin kinase(s) in G2/M phase lamin B phosphorylation.

    Funded by: NIGMS NIH HHS: GM 43186

    The Journal of biological chemistry 1994;269;29;19074-80

  • Dynamic properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase C beta.

    Gregorio CC, Repasky EA, Fowler VM and Black JD

    Department of Molecular Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263.

    Ankyrin is a well characterized membrane skeletal protein which has been implicated in the anchorage of specific integral membrane proteins to the spectrin-based membrane skeleton in a number of systems. In this study, the organization of ankyrin was examined in lymphocytes in relation to T cell function. Light and electron microscope immunolocalization studies revealed extensive heterogeneity in the subcellular distribution of ankyrin in murine tissue-derived lymphocytes. While ankyrin can be localized at the lymphocyte plasma membrane, it can also be accumulated at some distance from the cell periphery, in small patches or in a single discrete, nonmembrane-bound structure. Double immunofluorescence studies demonstrated that ankyrin colocalizes with spectrin and with the signal transducing molecule protein kinase C beta (PKC beta) in tissue-derived lymphocytes, suggesting a functional association between these molecules in the lymphocyte cytoplasm. In addition, T lymphocyte activation-related signals and phorbol ester treatment, both of which lead to PKC activation, cause a rapid translocation of ankyrin, together with spectrin and PKC beta, to a single Triton X-100-insoluble aggregate in the cytoplasm. This finding suggests a mechanism for the reported appearance of PKC in the particulate fraction of cells after activation: activated lymphocyte PKC beta may interact with insoluble cytoskeletal elements like ankyrin and spectrin. Further evidence for a link between the subcellular organization of these proteins and PKC activity is provided by the observation that inhibitors of PKC activity cause their concomitant redistribution to the cell periphery. The dynamic nature of lymphocyte ankyrin and its ability to accumulate at sites distant from the plasma membrane are properties which may be unique to the lymphocyte form of the molecule. Its colocalization with PKC beta in the lymphocyte cytoplasm, together with its redistribution in response to physiological signals, suggests that structural protein(s) may play a role in signal transduction pathways in this cell type. Our data support the conclusion that ankyrin is not solely involved in anchorage of proteins at the plasma membrane in lymphoid cells.

    Funded by: NCI NIH HHS: CA22786; NIAID NIH HHS: AI30131; NIGMS NIH HHS: GM34225

    The Journal of cell biology 1994;125;2;345-58

  • Human immunodeficiency virus-1 recombinant gp120 induces changes in protein kinase C isozymes--a preliminary report.

    Gupta S, Aggarwal S, Kim C and Gollapudi S

    Division of Basic and Clinical Immunology, University of California, Irvine 92717.

    Human immunodeficiency virus 1 (HIV-1) and its purified proteins activate target cell functions. Because protein kinase C (PKC) plays a crucial role in signal transduction and there is a molecular heterogeneity of PKC, we compared the effect of recombinant HIV-1 gp120 and phorbol ester (PMA) on PKC isozymes in monocytic U937 cells, with isozyme-specific antibodies using flow cytometry. All PKC isozymes except PKC-gamma were present in U937 cells. Both PMA and HIV-1 gp120 increased levels of calcium-dependent and -independent PKC isozymes. The most striking change was observed in PKC-zeta isozymes levels. This study for the first time demonstrates that HIV-1 gp120 affects calcium-independent PKC isozymes in U937 cells.

    International journal of immunopharmacology 1994;16;3;197-204

  • Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C.

    Blume-Jensen P, Siegbahn A, Stabel S, Heldin CH and Rönnstrand L

    Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.

    The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.

    The EMBO journal 1993;12;11;4199-209

  • Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain.

    Huang KP, Huang FL and Chen HC

    Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.

    A 7.5-kDa heat- and acid-stable rat brain protein kinase C (PKC) substrate was purified to near homogeneity by a two-step procedure using DEAE-cellulose and hydroxylapatite column chromatography. This 78-amino-acid protein has a sequence identical to that deduced from rat brain RC3 cDNA identified with a cortex-minus-cerebellum subtracted cDNA probe (J. B. Watson et al., J. Neurosci. Res. 26, 397-408, 1990) and exhibits extensive sequence identity to bovine brain neurogranin (J. Baudier et al., J. Biol. Chem. 266, 229-237, 1991). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis this protein, RC3, migrated as a M(r) 15-18K species in the presence of reducing agent and as heterogeneous species of M(r) 13-28K in the absence of reducing agent. Phosphorylation of RC3 by PKC alpha, beta, or gamma was stimulated by Ca2+, phospholipid, and diacylglycerol. A single site, Ser36, which is adjacent to the predicted calmodulin (CaM)-binding domain, was phosphorylated by these enzymes. Phosphorylation of RC3 by PKC or PKM, a protease-degraded PKC, was inhibited by CaM. The effect of CaM apparently targets at RC3, as phosphorylation of protamine sulfate by PKM was not inhibited by CaM. In the absence of Ca2+, RC3 formed a stoichiometric complex with CaM as evidenced by an increase in the M(r) determined by gel filtration chromatography. In the presence of Ca2+, the affinity of RC3 toward CaM is greatly reduced and Ca2+/CaM becomes less inhibitory of the PKM-catalyzed phosphorylation of RC3. Phosphorylation of RC3 by PKM prevented the interaction of this protein with CaM even in the absence of Ca2+. A 20-amino-acid synthetic peptide (AS-20F-W) containing the PKC phosphorylation site and CaM-binding domain of RC3 (Ala29-Ser48) with a substitution of Phe37 with tryptophan was used to monitor the interaction of this peptide with CaM by spectrofluorometry. In the absence of Ca2+, CaM caused negligible change in tryptophan fluorescence of the peptide; however, an enhancement and blue-shift of the emission fluorescence was observed in the presence of Ca2+. It seems that this synthetic peptide, as well as RC3 holoprotein, interacts with CaM through electrostatic interaction in the absence of Ca2+ but through hydrophobic interaction in the presence of Ca2+. In rat brain homogenate, RC3 formed a stable complex with CaM in the presence of Ca2+, as demonstrated by immunoblot analysis following gel filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

    Archives of biochemistry and biophysics 1993;305;2;570-80

  • Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.

    Zhang J, Wang L, Petrin J, Bishop WR and Bond RW

    Molecular Pharmacology Section, Schering-Plough Research Institute, Kenilworth, NJ 07033-0539.

    The autophosphorylation sites of the beta 2 isozyme of protein kinase C (PKC) were recently identified as Ser-16/Thr-17 near the NH2 terminus, Thr-314/Thr-324 in the hinge region, and Thr-634/Thr-641 near the COOH terminus [Flint, A.J., Paladini, R.D. & Koshland, D.E. (1990) Science 294, 408-411]. To define the role of autophosphorylation we constructed three site-directed mutants of PKC beta 1 isozyme in which each pair of phosphorylatable residues is changed to alanine. Wild-type PKC beta 1 and the mutant proteins were transiently overexpressed in COS cells, resulting in at least a 20-fold increase in [3H]phorbol 12,13-dibutyrate binding compared with control transfectants. Enzyme assays of PKC partially purified from transfected cells indicated at least a 5-fold increase in PKC activity upon expression of the wild-type protein or the NH2-terminal and hinge mutants. In contrast, no increased activity was detected upon expression of the COOH-terminal mutant. Immunoblot analysis using a beta isoform-specific antibody showed that wild-type, NH2-terminal mutant, and hinge mutant proteins are similarly distributed between the Triton-soluble and insoluble fractions. In contrast, the COOH-terminal mutant protein is largely Triton-insoluble. Immunoblot analysis also indicated that this mutant is resistant to down-regulation upon chronic exposure of cells to phorbol ester. Moreover, RNA blot analysis showed that overexpression of wild-type PKC but not of the COOH-terminal mutant enhances phorbol ester induction of c-FOS and c-JUN mRNA. Our results indicate that (i) alteration in the NH2-terminal and hinge autophosphorylation sites has no effect on PKC function by the criteria examined and (ii) the COOH-terminal autophosphorylation sites are critical for PKC function and possibly subcellular localization in COS cells.

    Proceedings of the National Academy of Sciences of the United States of America 1993;90;13;6130-4

  • Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

    Obeid LM, Blobe GC, Karolak LA and Hannun YA

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.

    The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

    Funded by: NHLBI NIH HHS: HL-43707

    The Journal of biological chemistry 1992;267;29;20804-10

  • Differential regulation of glycogen synthase kinase-3 beta by protein kinase C isotypes.

    Goode N, Hughes K, Woodgett JR and Parker PJ

    Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.

    In cells, stimulation of protein kinase C (PKC) results in the dephosphorylation of specific residues proximal to the DNA binding domain of c-Jun, a major component of the AP-1 transcription factor. Since phosphorylation of this region of c-Jun inhibits interaction with DNA, this pathway may contribute to PKC activation of AP-1. To determine the mechanism(s) underlying this pathway, possible interactions between PKC and proteins implicated in c-Jun regulation are being investigated. Here it is shown that glycogen synthase kinase-3 beta (GSK-3 beta), a serine/threonine kinase that specifically targets the inhibitory c-Jun phosphorylation sites, is phosphorylated in vitro by particular forms of PKC (alpha, beta 1, gamma greater than beta 2; not epsilon). By contrast, the related GSK-3 alpha is not a substrate for any of these PKC isotypes. Phosphorylation of GSK-3 beta by PKC results in its specific inactivation. These results are consistent with a model in which activation of PKC stimulates c-Jun DNA binding by inhibiting its phosphorylation by GSK-3 beta.

    The Journal of biological chemistry 1992;267;24;16878-82

  • Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

    Moss SJ, Doherty CA and Huganir RL

    Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)

    The Journal of biological chemistry 1992;267;20;14470-6

  • Down-regulation of human protein kinase C alpha is associated with terminal neutrophil differentiation.

    Devalia V, Thomas NS, Roberts PJ, Jones HM and Linch DC

    Department of Haematology, University College and Middlesex School of Medicine, London, England.

    We have established an RNase protection method to quantify the expression of mRNA for the human protein kinase C (PK-C) isoforms alpha, beta 1, beta 2, and gamma. This was used to investigate whether each isoform is differentially expressed during the differentiation of hematopoietic cells. Myeloid and lymphoid cells express PK-C alpha, beta 1, and beta 2 mRNAs in various proportions. PK-C gamma mRNA was detected in human brain, but not in hematopoietic cells. PK-C alpha mRNA decreases as HL-60 cells mature to a neutrophil phenotype in response to retinoic acid, but its abundance does not change during monocytic differentiation in response to vitamin D3. PK-C alpha mRNA and protein were undetectable in peripheral blood neutrophils, but are present in monocytes. The mRNAs for PK-C beta 1 and beta 2 isoforms increase during HL-60 differentiation and are expressed in both neutrophils and monocytes. Therefore, the PK-C alpha isoform is specifically down-regulated during human neutrophil terminal differentiation. These data suggest that mature neutrophil functions do not require the PK-C alpha isoform.

    Funded by: Wellcome Trust

    Blood 1992;80;1;68-76

  • Positive and negative regulation of the transcription of the human protein kinase C beta gene.

    Niino YS, Ohno S and Suzuki K

    Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.

    To analyze the mechanism of the cell type-specific expression of protein kinase C beta (PKC beta), we isolated the 5'-portion of the human gene for PKC beta and identified multiple positive and negative regulatory sequences that regulate its transcription. S1 nuclease mapping as well as primer extension analysis of the 5'-end of the PKC beta mRNA identified a putative transcriptional initiation site (position +1) 484 base pairs (bp) upstream of the first ATG codon. The 5'-upstream sequence contains a CCAAT sequence at position -110, but no TATA box. The transcriptional activities of various 5'-deletion mutants of the PKC beta gene upstream region, fused to the chloramphenicol acetyltransferase structural gene, were examined in terms of chloramphenicol acetyltransferase expression after transfection into three kinds of rodent cell lines: P19 and GH4C1, which are positive for the expression of PKC beta mRNA; and 3Y1, which is negative. Mutants containing a 5'-flanking sequence longer than 1.9 kilobases (kb) showed chloramphenicol acetyltransferase activities of the same order as the expression of the endogenous gene. This indicates that this region contains sequences regulating the cell-type specificity of PKC beta gene expression and that the specificity is determined at least partially at the level of transcription. The 1.9-kb sequence contains at least three positive elements: P1 (-56 to -234 bp), P2 (-234 to -411 bp), and PN (-1.4 to -1.9 kb). PN is active only in P19 cells, P1 in GH4C1 and P19 cells, and P2 in all three cell lines. In addition to these positive elements, there are negative elements: N1 (-411 to -674 bp), which is active in all three cell lines; and PN, which is active only in GH4C1 cells. These results suggest the presence of multiple trans-acting factors that act on these positive and negative cis-acting elements and regulate the cell type-specific expression of the PKC beta gene.

    The Journal of biological chemistry 1992;267;9;6158-63

  • A synthetic peptide with sequence identity to the transmembrane protein GP41 of HIV-1 inhibits distinct lymphocyte activation pathways dependent on protein kinase C and intracellular calcium influx.

    Ruegg CL and Strand M

    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    A synthetic peptide containing env amino acid (aa) sequence 581 to 597 of the transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) was tested for its effect on protein kinase C (PKC) and cytoplasmic free Ca2+ [( Ca2+]i) influx-dependent immune functions. We have previously shown that this peptide inhibits PKC-mediated phosphorylation and T-cell receptor-mediated [Ca2+]i influx as well as lymphoproliferation. In this study we demonstrate that the HIV-1 gp41 peptide aa581-597 inhibits lymphoproliferation stimulated via the distinct T-cell-activation molecules CD3, CD2, and CD28, as well as direct stimulation mediated by phorbol ester combined with ionomycin. Further, aa581-597 inhibits both PKC-dependent interleukin 2 (IL 2) production and the [Ca2+]i influx-dependent but PKC-independent induction of IL 2 receptor expression. The HIV-1 gp41 peptide also induces dramatic morphologic changes in lymphocytes, characterized by cytoplasmic ballooning and the acquisition of adherence to plastic, and these changes are dependent on both the length and the temperature of exposure. The results of this study suggest that the HIV-1 gp41 sequence aa581-597 acts at multiple sites to inhibit both PKC activity and [Ca2+]i influx, resulting in the abrogation of several distinct immune functions that are critical for an intact immune response and are defective in HIV-1-infected individuals.

    Funded by: NCI NIH HHS: CA-09243; NIAID NIH HHS: AI-28206

    Cellular immunology 1991;137;1;1-13

  • Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL-60 cell clones.

    McSwine-Kennick RL, McKeegan EM, Johnson MD and Morin MJ

    Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.

    In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.

    Funded by: NCI NIH HHS: CA-44589, T32 CA-09560

    The Journal of biological chemistry 1991;266;23;15135-43

  • Inhibition of protein kinase C and anti-CD3-induced Ca2+ influx in Jurkat T cells by a synthetic peptide with sequence identity to HIV-1 gp41.

    Ruegg CL and Strand M

    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

    We have previously shown that a synthetic peptide containing env residues 581-597 from HIV-1 inhibits lymphoproliferation of human PBMC. We have investigated the molecular mechanism(s) by which this HIV-1-derived peptide inhibits CD3-mediated signal transduction. We show that the peptide containing residues 581-597 from the HIV-1 transmembrane protein gp41 specifically inhibited the intracellular Ca2+ influx in Jurkat cells stimulated by the mAb OKT3 whereas it had no effect on the production of inositol triphosphate. In addition, the peptide inhibited protein kinase C (pkC)-mediated phosphorylation of the CD3 gamma-chain in intact cells and directly inhibited partially purified pkC. The inhibition was noncompetitive with respect to the substrates histone and ATP and independent of the regulatory domain of the enzyme. Furthermore, the peptide required internalization for inhibitory activity because no inhibition of lymphoproliferation was observed when cells were treated with peptide at 4 degrees C. Based on these results obtained with the peptide aa581-597, we postulate that the transmembrane protein gp41 of HIV-1 may inhibit pkC activity and thus block pkC-dependent immune function contributing to the immunosuppression of HIV-1-infected individuals.

    Funded by: NCI NIH HHS: CA-09243; NIAID NIH HHS: AI-28206

    Journal of immunology (Baltimore, Md. : 1950) 1990;144;10;3928-35

  • The phorbol ester TPA strongly inhibits HIV-1-induced syncytia formation but enhances virus production: possible involvement of protein kinase C pathway.

    Chowdhury IH, Koyanagi Y, Kobayashi S, Hamamoto Y, Yoshiyama H, Yoshida T and Yamamoto N

    Department of Virology and Parasitology, Yamaguchi University School of Medicine, Japan.

    Cocultivation of MOLT-4 and MOLT-4/HIVHTLV-IIIB cells with more than 0.01 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 hr strikingly inhibited HIV-induced syncytia formation resulting from cell to cell infection. Interestingly, the production of HIV-specific p24 antigen in the culture fluid was significantly enhanced by TPA. TPA down-modulated the expression of CD4. CD4 is essential for syncytia formation through interaction with viral envelope protein gp120 on the surface of MOLT-4 cells. The effects of TPA on syncytia formation and on CD4 expression were specifically interfered with by nontoxic doses of blockers of protein kinase C (PKC) such as staurosporine and H7. These data suggest that (1) TPA inhibits HIV-induced syncytia formation through down-modulation of CD4 molecules on the surface of MOLT-4 cells and (2) PKC may play an important role in cell to cell as well as in cell-free infection of HIV.

    Virology 1990;176;1;126-32

  • Trans-activation of HIV-1 LTR-directed gene expression by tat requires protein kinase C.

    Jakobovits A, Rosenthal A and Capon DJ

    Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080.

    Human immunodeficiency virus (HIV) spends a significant part of the viral life cycle as a latent provirus integrated into the host genome. Activation of latent HIV-1 requires mitogenic stimulation of the cell, which increases basal viral transcription, and the HIV-1 tat protein. As tat itself dramatically increases HIV-1 gene expression, it too is presumably regulated in the latent state, and may also be activated by mitogenic stimulation. We show here that depletion of protein kinase C (PKC), which is essential to the stimulation of T cells by several mitogens, dramatically reduces HIV-1 transactivation without affecting synthesis of tat protein. Transactivation in PKC-depleted cells can be restored by transfection with a PKC expression vector. The requirement for PKC in trans-activation does not involve the PMA-responsive enhancer elements responsible for the effect of mitogens on basal transcription. Our results indicate that PKC regulates the process of HIV-1 transactivation, suggesting a key role for the mitogenic induction of trans-activation in the transition of HIV from latency to productive growth.

    The EMBO journal 1990;9;4;1165-70

  • Identification of the protein kinase C phosphorylation site in neuromodulin.

    Apel ED, Byford MF, Au D, Walsh KA and Storm DR

    Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.

    Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NHLBI NIH HHS: HL-23606; NIGMS NIH HHS: GM-15731, GM-33708; ...

    Biochemistry 1990;29;9;2330-5

  • Assignment of human genes for phosphorylase kinase subunits alpha (PHKA) to Xq12-q13 and beta (PHKB) to 16q12-q13.

    Francke U, Darras BT, Zander NF and Kilimann MW

    Department of Human Genetics, Yale University School of Medicine, New Haven, CT.

    Phosphorylase kinase (PHK), the enzyme that activates glycogen phosphorylases in muscle, liver, and other tissues, is composed of four different subunits. Recently isolated rabbit muscle cDNAs for the larger two subunits, alpha and beta, have been used to map the location of their cognate sequences on human chromosomes. Southern blot analysis of rodent x human somatic cell hybrid panels, as well as in situ chromosomal hybridization, have provided evidence of single sites for both genes. The alpha subunit gene (PHKA) is located on the proximal long arm of the X chromosome in region Xq12-q13 near the locus for phosphoglycerate kinase (PGK1). X-linked mutations leading to PHK deficiency, known to exist in humans and mice, are likely to involve this locus. This hypothesis is consistent with the proximity of the Phk and Pgk-1 loci on the mouse X chromosome. In contrast, the beta subunit gene (PHKB) was found to be autosomal and was mapped to chromosome 16, region q12-q13 on the proximal long arm. Several different autosomally inherited forms of PHK deficiency for which the PHKB could be a candidate gene have been described in humans and rats.

    Funded by: NIGMS NIH HHS: GM26105

    American journal of human genetics 1989;45;2;276-82

  • Human immunodeficiency virus induces phosphorylation of its cell surface receptor.

    Fields AP, Bednarik DP, Hess A and May WS

    Johns Hopkins Oncology Center, Baltimore, Maryland 21231.

    AIDS is an immunoregulatory disorder characterized by depletion of the CD4+, helper/inducer lymphocyte population. The causative agent of this disease is the human immunodeficiency virus, HIV, which infects CD4+ cells and leads to cytopathic effects characterized by syncytia formation and cell death. Recent studies have demonstrated that binding of HIV to its cellular receptor CD4 is necessary for viral entry. We find that binding of HIV to CD4 induces rapid and sustained phosphorylation of CD4 which could involve protein kinase C. HIV-induced CD4 phosphorylation can be blocked by antibody against CD4 and monoclonal antibody against the HIV envelope glycoprotein gp120, indicating that a specific interaction between CD4 and gp120 is required for phosphorylation. Electron microscopy shows that a protein kinase C inhibitor does not impair binding of HIV to CD4+ cells, but causes an apparent accumulation of virus particles at the cell surface, at the same time inhibiting viral infectivity. These results indicate a possible role for HIV-induced CD4 phosphorylation in viral entry and identify a potential target for antiviral therapy.

    Nature 1988;333;6170;278-80

  • Primary structures of human protein kinase C beta I and beta II differ only in their C-terminal sequences.

    Kubo K, Ohno S and Suzuki K

    Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.

    Two types of cDNA clones encoding human protein kinase C (PKC) were isolated from a spleen cDNA library using rabbit protein kinase C beta I/beta II cDNA as a hybridization probe. Nucleotide sequence analyses of these cDNA inserts revealed complete primary structures of two distinct types of human protein kinase C beta I and beta II which differ only in their C-terminal 50 or 52 amino acid residues. It was concluded that there exist four distinct types of PKC, PKC alpha, beta I, beta II and gamma, in human as well as rabbit, and that the corresponding sequences are strictly conserved among mammalian species.

    FEBS letters 1987;223;1;138-42

  • Alternative splicing increases the diversity of the human protein kinase C family.

    Coussens L, Rhee L, Parker PJ and Ullrich A

    Department of Developmental Biology, Genentech, Inc., South San Francisco, CA 94080.

    Isolation of two protein kinase C (PKC) cDNA clones containing divergent carboxy-terminal sequences suggested a common genetic origin for these cDNAs. Partial characterization of the hPKC beta chromosomal gene provided direct evidence for the existence of two adjacent carboxy-terminal exons (beta 1 and beta 2) that are alternatively spliced to generate two types of hPKC beta sequences. PKC beta 1 and beta 2 mRNAs are expressed in a selective manner in both human hematopoietic cells and bovine brain tissues.

    DNA (Mary Ann Liebert, Inc.) 1987;6;5;389-94

  • Nucleotide sequence of the 3' portion of a human gene for protein kinase C beta I/beta II.

    Kubo K, Ohno S and Suzuki K

    Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.

    Nucleic acids research 1987;15;17;7179-80

  • Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways.

    Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield MD, Francke U and Ullrich A

    A new family of protein kinase C-related genes has been identified in bovine, human, and rat genomes. The alpha-, beta-, and gamma-type protein kinase sequences are highly homologous, include a kinase domain, and potential calcium-binding sites, and they contain interspersed variable regions. The corresponding genes are located on distinct human chromosomes; the possibility of even greater genetic complexity of this gene family is suggested by Northern and Southern hybridization analyses.

    Science (New York, N.Y.) 1986;233;4766;859-66

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000034 G2C Homo sapiens Pocklington H3 Human orthologues of cluster 3 (mouse) from Pocklington et al (2006) 30
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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