G2Cdb::Gene report

Gene id
G00000160
Gene symbol
Prkce (MGI)
Species
Mus musculus
Description
protein kinase C, epsilon
Orthologue
G00001409 (Homo sapiens)

Databases (11)

Curated Gene
OTTMUSG00000017512 (Vega mouse gene)
Gene
ENSMUSG00000045038 (Ensembl mouse gene)
18754 (Entrez Gene)
50 (G2Cdb plasticity & disease)
Gene Expression
NM_011104 (Allen Brain Atlas)
g02167 (BGEM)
18754 (Genepaint)
prkce (gensat)
Literature
176975 (OMIM)
Marker Symbol
MGI:97599 (MGI)
Protein Sequence
P16054 (UniProt)

Synonyms (3)

  • PKC[e]
  • PKCepsilon
  • Pkce

Literature (166)

Pubmed - other

  • Time-dependent effects of Prkce deletion on glucose homeostasis and hepatic lipid metabolism on dietary lipid oversupply in mice.

    Raddatz K, Turner N, Frangioudakis G, Liao BM, Pedersen DJ, Cantley J, Wilks D, Preston E, Hegarty BD, Leitges M, Raftery MJ, Biden TJ and Schmitz-Peiffer C

    Garvan Institute of Medical Research, 384 Victoria Street, Sydney, NSW 2010, Australia. k.raddatz@garvan.org.au

    We examined the time-dependent effects of deletion of the gene encoding protein kinase C epsilon (Prkce) on glucose homeostasis, insulin secretion and hepatic lipid metabolism in fat-fed mice.

    Methods: Prkce(-/-) and wild-type (WT) mice were fed a high-fat diet for 1 to 16 weeks and subjected to i.p. glucose tolerance tests (ipGTT) and indirect calorimetry. We also investigated gene expression and protein levels by RT-PCR, quantitative protein profiling (isobaric tag for relative and absolute quantification; iTRAQ) and immunoblotting. Lipid levels, mitochondrial oxidative capacity and lipid metabolism were assessed in liver and primary hepatocytes.

    Results: While fat-fed WT mice became glucose intolerant after 1 week, Prkce(-/-) mice exhibited normal glucose and insulin levels. iTRAQ suggested differences in lipid metabolism and oxidative phosphorylation between fat-fed WT and Prkce(-/-) animals. Liver triacylglycerols were increased in fat-fed Prkce(-/-) mice, resulting from altered lipid partitioning which promoted esterification of fatty acids in hepatocytes. In WT mice, fat feeding elevated oxygen consumption in vivo and in isolated liver mitochondria, but these increases were not seen in Prkce(-/-) mice. Prkce(-/-) hepatocytes also exhibited reduced production of reactive oxygen species (ROS) in the presence of palmitate. After 16 weeks of fat feeding, however, the improved glucose tolerance in fat-fed Prkce(-/-) mice was instead associated with increased insulin secretion during ipGTT, as we have previously reported.

    Prkce deletion ameliorates diet-induced glucose intolerance via two temporally distinct phenotypes. Protection against insulin resistance is associated with changes in hepatic lipid partitioning, which may reduce the acute inhibitory effects of fatty acid catabolism, such as ROS generation. In the longer term, enhancement of glucose-stimulated insulin secretion prevails.

    Diabetologia 2011;54;6;1447-56

  • Mechanism of lipid induced insulin resistance: activated PKCε is a key regulator.

    Dasgupta S, Bhattacharya S, Maitra S, Pal D, Majumdar SS, Datta A and Bhattacharya S

    Cellular and Molecular Endocrinology Laboratory, Department of Zoology, School of Life Science, Visva-Bharati University, Santiniketan 731235, India.

    Fatty acids (FAs) are known to impair insulin signaling in target cells. Accumulating evidences suggest that one of the major sites of FAs adverse effect is insulin receptor (IR). However, the underlying mechanism is yet unclear. An important clue was indicated in leptin receptor deficient (db/db) diabetic mice where increased circulatory FAs was coincided with phosphorylated PKCε and reduced IR expression. We report here that central to this mechanism is the phosphorylation of PKCε by FAs. Kinase dead mutant of PKCε did not augment FA induced IRβ downregulation indicating phosphorylation of PKCε is crucial for FA induced IRβ reduction. Investigation with insulin target cells showed that kinase independent phosphorylation of PKCε by FA occurred through palmitoylation. Mutation at cysteine 276 and 474 residues in PKCε suppressed this process indicating participation of these two residues in palmitoylation. Phosphorylation of PKCε endowed it the ability to migrate to the nuclear region of insulin target cells. It was intriguing to search about how translocation of phosphorylated PKCε occurred without having canonical nuclear localization signal (NLS). We found that F-actin recognized phospho-form of PKCε and chaperoned it to the nuclear region where it interact with HMGA1 and Sp1, the transcription regulator of IR and HMGA1 gene respectively and impaired HMGA1 function. This resulted in the attenuation of HMGA1 driven IR transcription that compromised insulin signaling and sensitivity.

    Biochimica et biophysica acta 2011;1812;4;495-506

  • Genetic ablation of PKC epsilon inhibits prostate cancer development and metastasis in transgenic mouse model of prostate adenocarcinoma.

    Hafeez BB, Zhong W, Weichert J, Dreckschmidt NE, Jamal MS and Verma AK

    Department of Human Oncology, Wisconsin Institute of Medical Research, Paul Carbone Comprehensive Cancer Center, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53792, USA.

    Protein kinase C epsilon (PKCε), a novel PKC isoform, is overexpressed in prostate cancer (PCa) and correlates with disease aggressiveness. However, the functional contribution of PKCε to development or progression of PCa remained to be determined. Here we present the first in vivo genetic evidence that PKCε is essential for both the development and metastasis of PCa in the transgenic mouse model of prostate adenocarcinoma (TRAMP). Heterozygous or homozygous genetic deletions of PKCε in FVB/N TRAMP inhibited PCa development and metastasis as analyzed by positron emission tomography/computed tomography, tumor weight determinations, and histopathology. We also examined biomarkers associated with tumor progression in this model, including markers of survival, proliferation, angiogenesis, inflammation, and metastatic progression. To find clues about the genes regulated by PKCε and linked to the Stat3 signaling pathway, we carried out focused PCR arrays of JAK/STAT signaling in excised PCa tissues from PKCε wild-type and nullizygous TRAMP mice. Notably, PKCε loss was associated with significant downregulation of proliferative and metastatic genes C/EBPβ (CCAAT/enhancer binding protein β), CRP (C-reactive protein), CMK, EGFR (epidermal growth factor receptor), CD64, Jun B, and gp130. Taken together, our findings offer the first genetic evidence of the role of PKCε in PCa development and metastasis. PKCε may be potential target for prevention and/or treatment of PCa.

    Funded by: NCI NIH HHS: 2 P30 CA014520-34, CA35368, R01 CA035368-26, R01 CA138761-03

    Cancer research 2011;71;6;2318-27

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • Transgenic overexpression of PKCε in the mouse prostate induces preneoplastic lesions.

    Benavides F, Blando J, Perez CJ, Garg R, Conti CJ, DiGiovanni J and Kazanietz MG

    Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA.

    It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCε, the product of the PRKCE gene, is up-regulated in various types of cancers including prostate, lung and breast cancer. To address a potential role for PKCs in prostate cancer progression we generated three mouse transgenic lines expressing PKCα, PKCδ, or PKCε in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCε and PB-PKCδ mice did not show any evident phenotype, PB-PKCε mice developed prostate hyperplasia as well as prostate intraepithelial neoplasia (PIN) that displayed enhanced phospho-Akt, phospho-S6, and phospho-Stat3 levels, as well as enhanced resistance to apoptotic stimuli. PKCε overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCε by adenoviral means in normal immortalized RWPE-1 prostate cells confers a growth advantage and hyperactivation of Erk and Akt. Our results argue for a causal link between PKCε overexpression and prostate cancer development.

    Funded by: NCI NIH HHS: P30 CA16672-30, R01 CA089202-10, R01-CA89202

    Cell cycle (Georgetown, Tex.) 2011;10;2;268-77

  • PKCε regulates contraction-stimulated GLUT4 traffic in skeletal muscle cells.

    Niu W, Bilan PJ, Yu J, Gao J, Boguslavsky S, Schertzer JD, Chu G, Yao Z and Klip A

    Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Immuno Microenvironment and Disease of the Educational Ministry of China, Tianjin Medical University, Tianjin, China. wniu@tijmu.edu.cn

    The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. Recently, we showed that carbachol, an acetylcholine analog, stimulates contraction of C2C12 myotube cultures and the rapid arrival of myc-epitope tagged GLUT4 glucose transporters at the cell surface. Here, we explore a role for protein kinase C (PKC) in regulating GLUT4 traffic. Cell surface carbachol-induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF-109203X, Gö6983, and Ro-31-8425 but not by the conventional PKC inhibitor Gö6976. C2C12 myotubes expressed several novel isoforms of PKC mRNA with PKCδ and PKCε in greater abundance. Carbachol stimulated phosphorylation of PKC isoforms and translocation of PKCδ and PKCε to membranes within 5 min. However, only a peptidic inhibitor of PKCε translocation (myristoylated-EAVSLKPT), but not one of PKCδ (myristoylated-SFNSYELGSL), prevented the GLUT4myc response to carbachol. Significant participation of PKCε in the carbachol-induced gain of GLUT4myc at the surface of C2C12 myotubes was further supported through siRNA-mediated PKCε protein knockdown. These findings support a role for novel PKC isoforms, especially PKCε, in contraction-stimulated GLUT4 traffic in muscle cells.

    Funded by: Canadian Institutes of Health Research: FRN-82420

    Journal of cellular physiology 2011;226;1;173-80

  • Differential expression of protein kinase C isoforms in coronary arteries of diabetic mice lacking the G-protein Gα11.

    Hoyer DP, Korkmaz Y, Grönke S, Addicks K, Wettschureck N, Offermanns S and Reuter H

    Department of Internal Medicine III, University of Cologne, Kerpener Str, 62, 50937 Cologne, Germany.

    Background: Diabetes mellitus counts as a major risk factor for developing atherosclerosis. The activation of protein kinase C (PKC) is commonly known to take a pivotal part in the pathogenesis of atherosclerosis, though the influence of specific PKC isozymes remains unclear. There is evidence from large clinical trials suggesting excessive neurohumoral stimulation, amongst other pathways leading to PKC activation, as a central mechanism in the pathogenesis of diabetic heart disease. The present study was therefore designed to determine the role of Gq-protein signalling via Gα11 in diabetes for the expression of PKC isozymes in the coronary vessels.

    Methods: The role of Gα11 in diabetes was examined in knockout mice with global deletion of Gα11 compared to wildtype controls. An experimental type 1-diabetes was induced in both groups by injection of streptozotocin. Expression and localization of the PKC isozymes α, βII, δ, ε, and ζ was examined by quantitative immunohistochemistry.

    Results: 8 weeks after induction of diabetes a diminished expression of PKC ε was observed in wildtype animals. This alteration was not seen in Gα11 knockout animals, however, these mice showed a diminished expression of PKCζ. Direct comparison of wildtype and knockout control animals revealed a diminished expression of PKC δ and ε in Gα11 knockout animals.

    Conclusion: The present study shows that expression of the nPKCs δ and ε in coronary vessels is under control of the g-protein Gα11. The reduced expression of PKC ζ that we observed in coronary arteries from Gα11-knockout mice compared to wildtype controls upon induction of diabetes could reduce apoptosis and promote plaque stability. These findings suggest a mechanism that may in part underlie the therapeutic benefit of RAS inhibition on cardiovascular endpoints in diabetic patients.

    Cardiovascular diabetology 2010;9;93

  • High-molecular weight hyaluronan reduced renal PKC activation in genetically diabetic mice.

    Campo GM, Avenoso A, Micali A, Nastasi G, Squadrito F, Altavilla D, Bitto A, Polito F, Rinaldi MG, Calatroni A, D'Ascola A and Campo S

    Department of Biochemical, Physiological and Nutritional Sciences, Section of Medical Chemistry, School of Medicine, University of Messina, Policlinico Universitario, Messina, Italy. gcampo@unime.it

    The cluster determinant (CD44) seems to play a key role in tissues injured by diabetes type 2. CD44 stimulation activates the protein kinase C (PKC) family which in turn activates the transcriptional nuclear factor kappa B (NF-κB) responsible for the expression of the inflammation mediators such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-18 (IL-18), inducible nitric oxide synthase (iNOS), and matrix metalloproteinases (MMPs). Regulation of CD44 interaction with its ligands depends greatly upon PKC. We investigated the effect of the treatment with high-molecular weight hyaluronan (HA) on diabetic nephropathy in genetically diabetic mice. BKS.Cg-m+/+Lepr(db) mice had elevated plasma insulin from 15 days of age and high blood sugar levels at 4 weeks. The severe nephropathy that developed was characterized by a marked increased in CD44 receptors, protein kinase C betaI, betaII, and epsilon (PKC(βI), PKC(βII), and PKCε) mRNA expression and the related protein products in kidney tissue. High levels of mRNA and related protein levels were also detected in the damaged kidney for NF-κB, TNF-α, IL-6, IL-18, MMP-7, and iNOS. Chronic daily administration of high-molecular mass HA for 2 weeks significantly reduced CD44, PKC(βI), PKC(βII), and PKCα gene expression and the related protein production in kidney tissue and TNF-α, IL-6, IL-18, MMP-7, and iNOS expression and levels also decreased. Histological analysis confirmed the biochemical data. However, blood parameters of diabetes were unchanged. These results suggest that the CD44 and PKC play an important role in diabetes and interaction of high-molecular weight HA with these proteins may reduce inflammation and secondary pathologies due to this disease.

    Biochimica et biophysica acta 2010;1802;11;1118-30

  • GABAA receptor trafficking is regulated by protein kinase C(epsilon) and the N-ethylmaleimide-sensitive factor.

    Chou WH, Wang D, McMahon T, Qi ZH, Song M, Zhang C, Shokat KM and Messing RO

    Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California 94688, USA.

    Disturbances in GABA(A) receptor trafficking contribute to several neurological and psychiatric disorders by altering inhibitory neurotransmission. Identifying mechanisms that regulate GABA(A) receptor trafficking could lead to better understanding of disease pathogenesis and treatment. Here, we show that protein kinase Cε (PKCε) regulates the N-ethylmaleimide-sensitive factor (NSF), an ATPase critical for membrane fusion events, and thereby promotes the trafficking of GABA(A) receptors. Activation of PKCε decreased cell surface expression of GABA(A) receptors and attenuated GABA(A) currents. Activated PKCε associated with NSF, phosphorylated NSF at serine 460 and threonine 461, and increased NSF ATPase activity, which was required for GABA(A) receptor downregulation. These findings identify new roles for NSF and PKCε in regulating synaptic inhibition through downregulation of GABA(A) receptors. Reducing NSF activity by inhibiting PKCε could help restore synaptic inhibition in disease states in which it is impaired.

    Funded by: NIAAA NIH HHS: AA013588, R01 AA013588, R01 AA013588-01, R37 AA013588

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;42;13955-65

  • Sigma-1 receptor-induced increase in murine spinal NR1 phosphorylation is mediated by the PKCalpha and epsilon, but not the PKCzeta, isoforms.

    Roh DH, Yoon SY, Seo HS, Kang SY, Moon JY, Song S, Beitz AJ and Lee JH

    Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

    Our previous studies have demonstrated that intrathecal (i.t.) administration of a sigma-1 receptor agonist facilitated peripheral nociception via calcium-dependent second messenger cascades including protein kinase C (PKC). We also showed that activation of spinal sigma-1 receptors increased the phosphorylation of the NMDA receptor NR1 subunit (pNR1) in the spinal cord dorsal horn, which resulted in the potentiation of NMDA receptor function. The present study was designed to examine the effect of different PKC isoform inhibitors on sigma-1 receptor-mediated pain facilitation and increased spinal pNR1 expression in mice. The intrathecal injection of the sigma-1 receptor agonist, PRE-084 (PRE, 3nmol/5mul) increased the frequency of paw withdrawal responses to mechanical stimuli (0.6g) and the number of spinal pNR1-immunoreactive (ir) cells. Intrathecal pretreatment with inhibitors (Go6976, PKCepsilonV1-2 or PKC zetapseudosubstrate) of the PKCalpha, epsilon or zeta isoforms significantly reduced the PRE-induced pain facilitatory effect. On the other hand, the PRE-induced increase in the number of spinal pNR1-ir neurons was only blocked by inhibitors of the PKCalpha and PKCepsilon isoforms, but not the PKCzeta isoform. These findings demonstrate that the sigma-1 receptor-induced increase in spinal pNR1 expression is mediated by the PKCalpha and PKC epsilon isoforms, which in turn contribute to the pain facilitation phenomenon. Conversely, the sigma-1 receptor activation of the PKCzeta isoform appears to be involved in a pain signaling pathway that is independent of spinal pNR1 modulation.

    Neuroscience letters 2010;477;2;95-9

  • Protein kinase C epsilon regulation of translocator protein (18 kDa) Tspo gene expression is mediated through a MAPK pathway targeting STAT3 and c-Jun transcription factors.

    Batarseh A, Li J and Papadopoulos V

    Department of Biochemistry and Molecular and Cell Biology, Georgetown University Medical Center, Washington, DC 20057, USA.

    Translocator protein TSPO is an 18 kDa protein implicated in numerous cell functions and is highly expressed in secretory and glandular tissues, especially in steroidogenic cells. TSPO expression is altered in pathological conditions such as certain cancers and neurological diseases. In search of the factors regulating Tspo expression, we recently showed that high levels of TSPO in steroidogenic cells may be due to high constitutive expression of protein kinase Cepsilon (PKCepsilon), while phorbol 12-myristate 13-acetate (PMA) activation of PKCepsilon drives inducible TSPO expression in nonsteroidogenic cells, likely through activator protein 1 (AP1). In this study, we aimed to identify the signal transduction pathway through which PKCepsilon regulates Tspo gene expression. The MEK1/2 specific inhibitor U0126, but not NFkappaB inhibitors, reduced basal Tspo promoter activity in TSPO-rich steroidogenic cells (MA-10 Leydig), as well as basal and PMA-induced Tspo promoter levels in TSPO-poor nonsteroidogenic cells (NIH-3T3 fibroblasts). AP1 and signal transducer and activation of transcription 3 (STAT3) have binding sites in the Tspo promoter and are downstream targets of PKCepsilon and MAPK (Raf-1-ERK1/2) pathways. PKCepsilon overexpression induced STAT3 phosphorylation in NIH-3T3 cells, while PKCepsilon knockdown reduced STAT3 and c-Jun phosphorylation in Leydig cells. MEK1/2, ERK2, c-Jun, and STAT3 knockdown reduced Tspo mRNA and protein levels in Leydig cells. Additionally, Raf-1 reduced Tspo mRNA levels in the same cells. MEK1/2, c-Jun, and STAT3 knockdown also reduced basal as well as PMA-induced Tspo mRNA levels in NIH-3T3 cells. Together, these results demonstrate that PKCepsilon regulates Tspo gene expression through a MAPK (Raf-1-MEK1/2-ERK1/2) signal transduction pathway, acting at least in part through c-Jun and STAT3 transcription factors.

    Funded by: NIEHS NIH HHS: R01 ES007747-15, R01 ES007747-16, R01 ES07747

    Biochemistry 2010;49;23;4766-78

  • Myocardial adenosine A(1)-receptor-mediated adenoprotection involves phospholipase C, PKC-epsilon, and p38 MAPK, but not HSP27.

    Fenton RA, Shea LG, Doddi C and Dobson JG

    Dept. of Physiology, Univ. of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655, USA. richard.fenton@umassmed.edu

    Adenosine via an adenosine A(1) receptor (A(1)R) is a negative feedback inhibitor of adrenergic stimulation in the heart, protecting it from toxic effects of overstimulation. Stimulation of the A(1)R results in the activation of G(i) protein, release of free Gbetagamma-subunits, and activation/translocation of PKC-epsilon to the receptor for activated C kinase 2 protein at the Z-line of the cardiomyocyte sarcomere. Using an anti-Gbetagamma peptide, we investigated the role of these subunits in the A(1)R stimulation of phospholipase C (PLC), with the premise that the resulting diacylglycerol provides for the activation of PKC-epsilon. Inositol 1,4,5-triphosphate release was an index of PLC activity. Chlorocyclopentyl adenosine (CCPA), an A(1)R agonist, increased inositol 1,4,5-triphosphate production by 273% in mouse heart homogenates, an effect absent in A(1)R knockout hearts and inhibited by anti-Gbetagamma peptide. In a second study, p38 MAPK and heat shock protein 27 (HSP27), found by others to be associated with the loss of myocardial contractile function, were postulated to play a role in the actions of A(1)R. Isoproterenol, a beta-adrenergic receptor agonist, increased the Ca(2+) transient and sarcomere shortening magnitudes by 36 and 49%, respectively. In the rat cardiomyocyte, CCPA significantly reduced these increases, an action blocked by the p38 MAPK inhibitor SB-203580. While CCPA significantly increased the phosphorylation of HSP27, this action was inhibited by isoproterenol. These data indicate that the activation of PKC-epsilon by A(1)R results from the activation of PLC via free Gbetagamma-subunits released upon A(1)R-induced dissociation of G(i)alphabetagamma. Attenuation of beta-adrenergic-induced contractile function by A(1)R may involve the activation of p38 MAPK, but not HSP27.

    Funded by: NHLBI NIH HHS: HL-84160

    American journal of physiology. Heart and circulatory physiology 2010;298;6;H1671-8

  • A role for PKCepsilon during C2C12 myogenic differentiation.

    Gaboardi GC, Ramazzotti G, Bavelloni A, Piazzi M, Fiume R, Billi AM, Matteucci A, Faenza I and Cocco L

    Cellular Signalling Laboratory, Department of Human Anatomical Sciences, University of Bologna, 40126 Bologna, Italy.

    In a previous report we have demonstrated that PLCgamma1 is involved in the differentiation process of C2C12 myoblasts, induced by insulin administration. In order to identify the downstream targets of PLCgamma1-dependent signalling, we have analyzed the expression of DAG-dependent PKC isoforms during muscle differentiation. We show that during myotube formation, there is a marked increase of PKCepsilon and eta expression, and that PKCepsilon is able to form a complex with PLCgamma1. The increase in PKCepsilon amount during myogenic differentiation is associated to an increase in PKCepsilon activity as well. Immunofluorescence analysis indicated that in growing C2C12 cells both PLCgamma1 and PKCepsilon localize in the cytoplasm with a distinct perinuclear accumulation. In insulin-treated cells, the expression of PLCgamma1 and PKCepsilon increases and the two proteins are still distributed mainly in the perinuclear region of the myotubes. We show that PLCgamma1-PKCepsilon complex co-localizes with protein 58K, a specific Golgi marker. Moreover, our results indicate that the Golgi-associated PKCepsilon form, i.e. PKCepsilon phosphorylated at Ser 729, is increased in differentiated myoblasts. Since it has been previously demonstrated that in C2C12 cells after insulin administration cyclin D3 levels could be modulated by PLCgamma1, we analyzed the effect on cyclin D3 expression of either PKCepsilon overexpression or silencing, in order to investigate whether PKCepsilon could also affect cyclin D3 expression. The results showed that either a modification of PKCepsilon expression or a change in its catalytic activity determines a variation of cyclin D3 levels and muscle differentiation in terms of myogenin expression. These data support a role for PKCepsilon in regulating insulin inositide-dependent PLCgamma1 signalling in skeletal muscle differentiation.

    Cellular signalling 2010;22;4;629-35

  • Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCvarepsilon) knockout mice.

    Budas GR, Disatnik MH, Chen CH and Mochly-Rosen D

    Department of Chemical and Systems Biology, Stanford University School of Medicine, CCSR, Rm 3145A, 269 Campus Drive, Stanford, CA 94305-5174, USA.

    Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCvarepsilon). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCvarepsilon substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCvarepsilon knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCvarepsilon and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCvarepsilon, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCvarepsilon knockout hearts. Our data suggest that ALDH2 is downstream of PKCvarepsilon in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCvarepsilon to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.

    Funded by: NIAAA NIH HHS: AA11147, R01 AA011147, R01 AA011147-13, R37 AA011147

    Journal of molecular and cellular cardiology 2010;48;4;757-64

  • Interleukin-1 Receptor-Associated Kinase-1 (IRAK-1) functionally associates with PKCepsilon and VASP in the regulation of macrophage migration.

    Gan L and Li L

    Department of Biology, Virginia Tech, Blacksburg, VA 24061, USA.

    Macrophage migration is mediated by complex cellular signaling processes and cytoskeleton re-arrangement. In particular, recent advances indicate that the innate immunity signaling process plays a key role in the regulation of macrophage migration. In this report, we have provided evidence demonstrating the involvement of a key innate immunity signaling kinase, Interleukin-1 Receptor-Associated Kinase-1 (IRAK-1) as a critical modulator of macrophage migration. Macrophage migration induced by phorbol 12-myristate 13-acetate (PMA) is significantly attenuated in IRAK-1(-/-) macrophages as compared to wild type macrophages. Mechanistically, we demonstrated that IRAK-1 works downstream of PKCepsilon and upstream of VASP, a member of Ena/VASP family proteins. IRAK-1 forms a close complex with PKCepsilon as well as VASP, and participates in PMA-induced phosphorylation of VASP. Notably, IRAK-1 contains a novel EVH1 domain binding motif (L(167)WPPPP) within its N-terminus, which is responsible for its interaction with VASP. The mutant IRAK-1 (L167A/W168A) fails to associate with VASP. Our findings provide a novel facet regarding the molecular signaling process regulating macrophage migration.

    Funded by: NIAID NIH HHS: R01 AI064414, R01 AI064414-06

    Molecular immunology 2010;47;6;1278-82

  • PKCepsilon overexpression, irrespective of genetic background, sensitizes skin to UVR-induced development of squamous-cell carcinomas.

    Sand JM, Aziz MH, Dreckschmidt NE, Havighurst TC, Kim K, Oberley TD and Verma AK

    Department of Human Oncology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.

    Chronic exposure to UVR is the major etiologic factor in the development of human skin cancers including squamous-cell carcinoma (SCC). We have previously shown that protein Kinase C epsilon (PKCepsilon) transgenic mice on FVB/N background, which overexpress PKCepsilon protein approximately eightfold over endogenous levels in epidermis, exhibit about threefold more sensitivity than wild-type littermates to UVR-induced development of SCC. To determine whether it is PKCepsilon and not the mouse genetic background that determines susceptibility to UVR carcinogenesis, we cross-bred PKCepsilon FVB/N transgenic mice with SKH-1 hairless mice to generate PKCepsilon-overexpressing SKH-1 hairless mice. To evaluate the susceptibility of PKCepsilon SKH-1 hairless transgenic mice to UVR carcinogenesis, the mice were exposed to UVR (1-2 KJ m(-2)) three times weekly from a bank of six kodacel-filtered FS40 sunlamps. As compared with the wild-type hairless mice, PKCepsilon overexpression in SKH-1 hairless mice decreased the latency (12 weeks), whereas it increased the incidence (twofold) and multiplicity (fourfold) of SCC. The SKH hairless transgenic mice were observed to be as sensitive as FVB/N transgenic mice to UVR-induced development of SCC and expression of proliferative markers (proliferating cell nuclear antigen, signal transducers and activators of transcription 3, and extracellular signal-regulated kinase 1/2). The results indicate that PKCepsilon level dictates susceptibility, irrespective of genetic background, to UVR carcinogenesis.

    Funded by: NCI NIH HHS: CA 35368, R01 CA035368-14S1, R01 CA035368-15, R01 CA035368-15S1, R01 CA035368-16, R01 CA035368-16S1, R01 CA035368-17A2, R01 CA035368-18, R01 CA035368-19, R01 CA035368-20, R01 CA035368-21, R01 CA035368-22A1, R01 CA035368-23, R01 CA035368-24; NIEHS NIH HHS: T32 ES007015-22, T32 ES007015-23, T32 ES007015-24, T32 ES007015-25, T32 ES007015-26, T32 ES007015-27, T32 ES007015-32, T32ES007015

    The Journal of investigative dermatology 2010;130;1;270-7

  • Diverse roles for protein kinase C delta and protein kinase C epsilon in the generation of high-fat-diet-induced glucose intolerance in mice: regulation of lipogenesis by protein kinase C delta.

    Frangioudakis G, Burchfield JG, Narasimhan S, Cooney GJ, Leitges M, Biden TJ and Schmitz-Peiffer C

    Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales, 2010, Australia.

    This study aimed to determine whether protein kinase C (PKC) delta plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCepsilon in the generation of insulin resistance in skeletal muscle.

    Methods: Prkcd (-/-), Prkce (-/-) and wild-type mice were fed high-fat diets and subjected to glucose tolerance tests. Blood glucose levels and insulin responses were determined during the tests. Insulin signalling in liver and muscle was assessed after acute in vivo insulin stimulation by immunoblotting with phospho-specific antibodies. Activation of PKC isoforms in muscle from Prkce (-/-) mice was assessed by determining intracellular distribution. Tissues and plasma were assayed for triacylglycerol accumulation, and hepatic production of lipogenic enzymes was determined by immunoblotting.

    Results: Both Prkcd (-/-) and Prkce (-/-) mice were protected against high-fat-diet-induced glucose intolerance. In Prkce (-/-) mice this was mediated through enhanced insulin availability, while in Prkcd (-/-) mice the reversal occurred in the absence of elevated insulin. Neither the high-fat diet nor Prkcd deletion affected maximal insulin signalling. The activation of PKCdelta in muscle from fat-fed mice was enhanced by Prkce deletion. PKCdelta-deficient mice exhibited reduced liver triacylglycerol accumulation and diminished production of lipogenic enzymes.

    Deletion of genes encoding isoforms of PKC can improve glucose intolerance, either by enhancing insulin availability in the case of Prkce, or by reducing lipid accumulation in the case of Prkcd. The absence of PKCepsilon in muscle may be compensated by increased activation of PKCdelta in fat-fed mice, suggesting that an additional role for PKCepsilon in this tissue is masked.

    Diabetologia 2009;52;12;2616-20

  • Opposite effects of protein kinase C beta1 (PKCbeta1) and PKCepsilon in the metastatic potential of a breast cancer murine model.

    Grossoni VC, Todaro LB, Kazanietz MG, de Kier Joffé ED and Urtreger AJ

    Research Area, Institute of Oncology Angel H. Roffo, University of Buenos Aires, Av. San Martín 5481, (C1417DTB), Buenos Aires, Argentina.

    In this paper we investigated whether protein kinase C (PKC) beta1 and PKCepsilon, members of the classical and novel PKC family, respectively, induce phenotypic alterations that could be associated with tumor progression and metastatic dissemination in a murine model of breast cancer. Stable overexpression of PKCbeta1 in LM3 cells altered their ability to proliferate, adhere, and survive, and impaired their tumorigenicity and metastatic capacity. Moreover, PKCbeta1 induced the re-expression of fibronectin, an extracellular matrix glycoprotein which loss has been associated with the acquisition of a transformed phenotype in different cell models, and exerted an important inhibition on proteases production, effects that probably impact on LM3 invasiveness and dissemination. Conversely, PKCepsilon overexpression enhanced LM3 survival, anchorage-independent growth, and caused a significant increase in spontaneous lung metastasis. Our results suggest PKCbeta1 functions as an inhibitory protein for tumor growth and metastasis dissemination whereas PKCepsilon drives metastatic dissemination without affecting primary tumor growth.

    Breast cancer research and treatment 2009;118;3;469-80

  • Selective activation of protein kinase C-delta and -epsilon by 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U).

    Coutinho I, Pereira G, Simões MF, Côrte-Real M, Gonçalves J and Saraiva L

    Laboratório de Microbiologia, REQUIMTE/CEQUP, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4050-047 Porto, Portugal.

    6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U) is a diterpene compound isolated from Plectranthus grandidentatus with an antiproliferative effect on several human cancer cell lines. Herein, we studied the modulatory activity of coleon U on individual isoforms of the three protein kinase C (PKC) subfamilies, classical (cPKC-alpha and -betaI), novel (nPKC-delta and -epsilon) and atypical (aPKC-zeta), using a yeast PKC assay. The results showed that, whereas the PKC activator phorbol-12-myristate-13-acetate (PMA) activated every PKC tested except aPKC, coleon U had no effect on aPKC and cPKCs. Besides, the effect of coleon U on nPKCs was higher than that of PMA. This revealed that coleon U was a potent and selective activator of nPKCs. The isoform-selectivity of coleon U for nPKC-delta and -epsilon was confirmed using an in vitro PKC assay. Most importantly, while PMA activated nPKCs inducing an isoform translocation from the cytosol to the plasma membrane and a G2/M cell cycle arrest, coleon U induced nPKCs translocation to the nucleus and a metacaspase- and mitochondrial-dependent apoptosis. This work therefore reconstitutes in yeast distinct subcellular translocations of a PKC isoform and the subsequent distinct cellular responses reported for mammalian cells. Together, our study identifies a new isoform-selective PKC activator with promising pharmacological applications. Indeed, since coleon U has no effect on cPKCs and aPKC, recognised as anti-apoptotic proteins, and selectively induces an apoptotic pathway dependent on nPKC-delta and -epsilon activation, it represents a promising compound for evaluation as an anti-cancer drug.

    Biochemical pharmacology 2009;78;5;449-59

  • Deletion of PKCepsilon selectively enhances the amplifying pathways of glucose-stimulated insulin secretion via increased lipolysis in mouse beta-cells.

    Cantley J, Burchfield JG, Pearson GL, Schmitz-Peiffer C, Leitges M and Biden TJ

    Diabetes and Obesity Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

    Objective: Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of beta-cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase C epsilon (PKCepsilon) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCepsilon.

    We cultured islets isolated from PKCepsilon knockout (PKCepsilonKO) mice in palmitate prior to measuring GSIS, Ca(2+) responses, palmitate esterification products, lipolysis, lipase activity, and gene expression.

    Results: The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated Ca(2+) influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCepsilonKO beta-cells was revealed under conditions in which functional ATP-sensitive K(+) channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCepsilonKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCepsilonKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCepsilon deletion.

    Conclusions: Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine beta-cells, and they suggest an interaction between PKCepsilon and lipolysis. These results further highlight the therapeutic potential of PKCepsilon inhibition to enhance GSIS from the beta-cell under conditions of lipid excess.

    Diabetes 2009;58;8;1826-34

  • Protein kinase Cepsilon inhibits UVR-induced expression of FADD, an adaptor protein, linked to both Fas- and TNFR1-mediated apoptosis.

    Aziz MH, Sundling KE, Dreckschmidt NE and Verma AK

    Department of Human Oncology, Medical School, University of Wisconsin, Madison, Wisconsin 53792, USA.

    Protein kinase C (PKC)epsilon overexpression in FVB/N transgenic mice sensitized skin to UVR-induced development of squamous cell carcinomas and suppressed formation of sunburn cells, which are DNA-damaged keratinocytes undergoing apoptosis. Here, we elucidated the mechanisms associated with the inhibition of UVR-induced appearance of sunburn cells in PKCepsilon transgenic mice. We found that the inhibition of UVR-induced sunburn cell formation in PKCepsilon transgenic mice may be the result of the inhibition of the expression of Fas, Fas ligand, and the mammalian death adaptor protein termed Fas-associated with death domain (FADD). The adaptor protein FADD is the key component of the death-inducing signaling complex of both Fas and tumor necrosis factor receptor 1. A decreased expression of epidermal FADD was observed after a single UVR exposure. However, a complete loss of FADD expression was found after four (Monday, Wednesday, Friday, and Monday) repeated UVR exposures. FADD transmits apoptotic signals from death receptors to the downstream initiator caspase-8 and connects to the mitochondrial intrinsic apoptotic signal transduction pathway by the cleavage of Bid, a Bcl-2 family member. PKCepsilon-mediated loss of FADD expression inhibited UVR signals to the activation of both extrinsic and intrinsic apoptotic pathways.

    Funded by: NCI NIH HHS: CA102431, CA35368, R01 CA035368, R01 CA035368-14S1, R01 CA035368-15, R01 CA035368-15S1, R01 CA035368-16, R01 CA035368-16S1, R01 CA035368-17A2, R01 CA035368-18, R01 CA035368-19, R01 CA035368-20, R01 CA035368-21, R01 CA035368-22A1, R01 CA035368-23, R01 CA035368-24, R01 CA102431, R01 CA102431-01A1, R01 CA102431-02, R01 CA102431-03, R01 CA102431-04, R01 CA102431-05; NIGMS NIH HHS: T32 GM008692

    The Journal of investigative dermatology 2009;129;8;2011-21

  • PKC epsilon has an alcohol-binding site in its second cysteine-rich regulatory domain.

    Das J, Pany S, Rahman GM and Slater SJ

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, USA. jdas@uh.edu

    Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that an alcohol-binding site is present in PKC alpha in its C1 domain, which consists of two cysteine-rich subdomains, C1A and C1B. A PKC epsilon-knockout mouse showed a significant decrease in alcohol consumption compared with the wild-type. The aim of the present study was to investigate whether an alcohol-binding site could be present in PKC epsilon. Here we show that ethanol inhibited PKC epsilon activity in a concentration-dependent manner with an EC50 (equilibrium ligand concentration at half-maximum effect) of 43 mM. Ethanol, butanol and octanol increased the binding affinity of a fluorescent phorbol ester SAPD (sapintoxin-D) to PKC epsilon C1B in a concentration-dependent manner with EC50 values of 78 mM, 8 mM and 340 microM respectively, suggesting the presence of an allosteric alcohol-binding site in this subdomain. To identify this site, PKC epsilon C1B was photolabelled with 3-azibutanol and 3-azioctanol and analysed by MS. Whereas azibutanol preferentially labelled His236, Tyr238 was the preferred site for azioctanol. Inspection of the model structure of PKC epsilon C1B reveals that these residues are 3.46 A (1 A=0.1 nm) apart from each other and form a groove where His236 is surface-exposed and Tyr238 is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding in terms of both photolabelling and alcohol-induced SAPD binding in the mutant H236A/Y238A. Whereas Tyr238 was labelled in mutant H236A, His236 was labelled in mutant Y238A. The present results provide direct evidence for the presence of an allosteric alcohol-binding site on protein kinase C epsilon and underscore the role of His236 and Tyr238 residues in alcohol binding.

    Funded by: PHS HHS: G096452

    The Biochemical journal 2009;421;3;405-13

  • Amygdala protein kinase C epsilon controls alcohol consumption.

    Lesscher HM, Wallace MJ, Zeng L, Wang V, Deitchman JK, McMahon T, Messing RO and Newton PM

    Department of Neurology, Ernest Gallo Clinic and Research Center, University of California at San Francisco, Emeryville, CA, USA. H.M.B.Lesscher@umcutrecht.nl

    Alcoholism is a progressive disorder that involves the amygdala. Mice lacking protein kinase C epsilon (PKCepsilon) show reduced ethanol consumption, sensitivity and reward. We therefore investigated whether PKCepsilon signaling in the amygdala is involved in ethanol consumption. Local knockdown of PKCepsilon in the amygdala reduced ethanol consumption and preference in a limited-access paradigm. Further, mice that are heterozygous for the PKCepsilon allele consume less ethanol compared with wild-type mice in this paradigm. These mice have a >50% reduction in the abundance of PKCepsilon in the amygdala compared with wild-type mice. We conclude that amygdala PKCepsilon is important for ethanol consumption in mice.

    Funded by: NIAAA NIH HHS: AA013588, R37 AA013588-07

    Genes, brain, and behavior 2009;8;5;493-9

  • Protein kinase C-epsilon regulates local calcium signaling in airway smooth muscle cells.

    Liu QH, Zheng YM, Korde AS, Li XQ, Ma J, Takeshima H and Wang YX

    Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.

    Protein kinase C (PKC) is known to regulate ryanodine receptor (RyR)-mediated local Ca(2+) signaling (Ca(2+) spark) in airway and vascular smooth muscle cells (SMCs), but its specific molecular mechanisms and functions still remain elusive. In this study, we reveal that, in airway SMCs, specific PKCepsilon peptide inhibitor and gene deletion significantly increased the frequency of Ca(2+) sparks, and decreased the amplitude of Ca(2+) sparks in the presence of xestospogin-C to eliminate functional inositol 1,4,5-triphosphate receptors. PKCepsilon activation with phorbol-12-myristate-13-acetate significantly decreased Ca(2+) spark frequency and increased Ca(2+) spark amplitude. The effect of PKCepsilon inhibition or activation on Ca(2+) sparks was completely lost in PKCepsilon(-/-) cells. PKCepsilon inhibition or PKCepsilon activation was unable to affect Ca(2+) sparks in RyR1(-/-) and RyR1(+/-) cells. Modification of RyR2 activity by FK506-binding protein 12.6 homozygous or RyR2 heterozygous gene deletion did not prevent the effect of PKCepsilon inhibition or activation. RyR3 homogenous gene deletion did not block the effect of PKCepsilon inhibition and activation, either. PKCepsilon inhibition promotes agonist-induced airway muscle contraction, whereas PKCepsilon activation produces an opposite effect. Taken together, these results indicate that PKCepsilon regulates Ca(2+) sparks by specifically interacting with RyR1, which plays an important role in the control of contractile responses in airway SMCs.

    Funded by: NHLBI NIH HHS: R01HL071000

    American journal of respiratory cell and molecular biology 2009;40;6;663-71

  • Role of protein kinase C and phosphoinositide 3-kinase-Akt in substance P-induced proinflammatory pathways in mouse macrophages.

    Sun J, Ramnath RD, Tamizhselvi R and Bhatia M

    Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    Neuropeptide modulation of immune cell function is an important mechanism of neuro-immune intersystem crosstalk. Substance P (SP) is one such key neuropeptide involved. In this study, we investigated the yet unexplored cellular mechanisms of SP-mediated inflammatory responses in macrophages using a mouse macrophage-like cell line RAW 264.7 and isolated peritoneal macrophages. We found that the conventional PKCalpha and novel PKCdelta and epsilon were selectively activated by SP via its primary neurokinin-1 receptor (NK-1R) on the cells. Activation of these PKC isoforms mediated the activation of downstream extracellular signal-regulated kinase-1/2 (ERK1/2) and the transcription factor NF-kappaB, which drove the transcription of inducible chemokines in macrophages. Additionally, phosphoinositide 3-kinase (PI3K)-Akt was also activated by SP/NK-1R in macrophages. Inhibition of PI3K-Akt pathway attenuated ERK1/2 and NF-kappaB activation, suggesting it also played a part in SP-induced cellular inflammatory response. Kinetic analysis indicated that PKC isoforms induced early ERK1/2 activation, while PI3K-Akt contributed to the pathway at later time points. It was further demonstrated that PKC and PI3K-Akt were activated independent of each other. Collectively, our results suggest that SP/NK-1R activates two convergent proinflammatory signaling pathways, PKCs and PI3K-Akt, resulting in ERK1/2 and NF-kappaB activation and chemokine production in mouse macrophages.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009;23;4;997-1010

  • Hypoxia-regulated activity of PKCepsilon in the lens.

    Akoyev V, Das S, Jena S, Grauer L and Takemoto DJ

    Department of Biochemistry, Kansas State University, Manhattan, Kansas.

    Purpose: To show that hypoxia is necessary to prevent opacification of the lens. Protein kinase C (PKC)-epsilon serves a role that is distinct from PKC-gamma when both PKC isoforms are expressed in the lens. PKCepsilon serves a very important role in hypoxic conditions, helping to prevent opacification of the lens.

    Methods: Digital image analysis, confocal microscopy, dye transfer assay, coimmunoprecipitation, Western blot analysis, and enzyme activity assays were used, respectively, to study opacification of the lens, intercellular communications, cellular localization of connexin-43 (Cx43), and the interactions between PKCepsilon, PKCgamma, and Cx43 in the lens epithelial cells.

    Results: Hypoxic conditions (1%-5% of oxygen) were very important in maintaining clarity of the lenses of wild-type (WT) mice. Normoxic conditions induced opacification of the WT lens. Lenses from the PKCepsilon-knockout mice underwent rapid opacification, even in hypoxic conditions. Hypoxia did not induce apoptosis in the lens epithelial cells, judging by the absence of active caspase-3, and it did not change intercellular communication and did not affect the number and localization of junctional Cx43 plaques in the lens epithelial cell culture. Hypoxia activated PKCepsilon, whereas phorbol ester (TPA), oxidation (H(2)O(2)), and insulin-like growth factor-1 (IGF-1) activated PKCgamma and decreased the activity of PKCepsilon. Hypoxia did not induce the phosphorylation of the Cx43.

    Conclusions: Hypoxia-induced activation of PKCepsilon is very important in surviving hypoxia and maintaining the clarity of the lens. However, PKCgamma is utilized in the control of Cx43 gap junctions.

    Funded by: NCRR NIH HHS: P20 RR016475; NEI NIH HHS: EY0R01-13421

    Investigative ophthalmology & visual science 2009;50;3;1271-82

  • PKCepsilon plays a causal role in acute ethanol-induced steatosis.

    Kaiser JP, Beier JI, Zhang J, David Hoetker J, von Montfort C, Guo L, Zheng Y, Monia BP, Bhatnagar A and Arteel GE

    Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Louisville, KY 40292, USA.

    Steatosis is a critical stage in the pathology of alcoholic liver disease (ALD), and preventing steatosis could protect against later stages of ALD. PKCepsilon has been shown to contribute to hepatic steatosis in experimental non-alcoholic fatty liver disease (NAFLD); however, the role of PKCepsilon in ethanol-induced steatosis has not been determined. The purpose of this study was to therefore test the hypothesis that PKCepsilon contributes to ethanol-induced steatosis. Accordingly, the effect of acute ethanol on indices of hepatic steatosis and insulin signaling were determined in PKCepsilon knockout mice and in wild-type mice that received an anti-sense oligonucleotide (ASO) to knockdown PKCepsilon expression. Acute ethanol (6g/kg i.g.) caused a robust increase in hepatic non-esterified free fatty acids (NEFA), which peaked 1h after ethanol exposure. This increase in NEFA was followed by elevated diacylglycerols (DAG), as well as by the concomitant activation of PKCepsilon. Acute ethanol also changed the expression of insulin-responsive genes (i.e. increased G6Pase, downregulated GK), in a pattern indicative of impaired insulin signaling. Acute ethanol exposure subsequently caused a robust increase in hepatic triglycerides. The accumulation of triglycerides caused by ethanol was blunted in ASO-treated or in PKCepsilon(-/-) mice. Taken together, these data suggest that the increase in NEFA caused by hepatic ethanol metabolism leads to an increase in DAG production via the triacylglycerol pathway. DAG then subsequently activates PKCepsilon, which then exacerbates hepatic lipid accumulation by inducing insulin resistance. These data also suggest that PKCepsilon plays a causal role in at least the early phases of ethanol-induced liver injury.

    Funded by: NIAAA NIH HHS: AA 003624, F31 AA 017346, R01 AA003624-27A1, R01 AA003624-28, R01 AA003624-29; NIEHS NIH HHS: ES 11860

    Archives of biochemistry and biophysics 2009;482;1-2;104-11

  • Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II.

    Oestreich EA, Malik S, Goonasekera SA, Blaxall BC, Kelley GG, Dirksen RT and Smrcka AV

    Department of Pharmacology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

    Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.

    Funded by: NIAMS NIH HHS: AR044657, R01 AR044657, R01 AR044657-12; NIDDK NIH HHS: DK56294; NIGMS NIH HHS: R01 GM053536, R01 GM053536-10; PHS HHS: GMR01053536

    The Journal of biological chemistry 2009;284;3;1514-22

  • Phorbol-12-myristate 13-acetate acting through protein kinase Cepsilon induces translocator protein (18-kDa) TSPO gene expression.

    Batarseh A, Giatzakis C and Papadopoulos V

    Department of Biochemistry & Molecular and Cell Biology, Georgetown University Medical Center, Washington, DC 20057, USA.

    Translocator protein (TSPO) is an 18-kDa cholesterol-binding protein that is expressed at high levels in steroid synthesizing and several cancer cells where it is involved in steroidogenesis and cell proliferation, respectively. The factors regulating Tspo expression are unknown. We analyzed Tspo transcriptional responses to the tumor promoter, phorbol-12-myristate 13-acetate (PMA), in cells with varying TSPO levels. PMA induced Tspo promoter activity and Tspo mRNA levels in TSPO-poor nonsteroidogenic cells (NIH-3T3 fibroblasts and COS-7 kidney) but not in TSPO-rich steroidogenic cells (MA-10 Leydig) with high basal Tspo transcriptional activity. The stimulatory effect of PMA was mediated by an 805-515-bp region upstream of the transcription start site. Electrophoretic mobility shift assay (EMSA) revealed that PMA induced binding of c-jun and GA-binding protein transcription factor (GABP-alpha) to their respective activator protein 1 (AP1) and v-ets erythroblastosis virus E26 oncogene homologue (Ets) sites in this region. Protein kinase C (PKC)-specific inhibitors blocked PMA induction of Tspo promoter activity with an inhibition profile suggestive of involvement of PKCepsilon. PKCepsilon expression correlated with TSPO content in the three cell lines. In NIH-3T3 cells, PKCepsilon overexpression induced Tspo promoter activity and mRNA levels and enhanced PMA-induced up regulation of c-jun and TSPO. In MA-10 cells, a PKCepsilon-specific translocation inhibitor peptide reduced basal Tspo promoter activity. PKCepsilon siRNA pool reduced PKCepsilon and TSPO levels in MA-10 cells indicating a role for PKCepsilon in regulating TSPO expression. Taken together, these data suggest that elevated TSPO expression in steroidogenic cells may be due to high constitutive expression of PKCepsilon that renders them unresponsive to further induction while PMA activation of PKCepsilon drives inducible TSPO expression in nonsteroidogenic cells, likely through AP1 and Ets.

    Funded by: NIEHS NIH HHS: R01 ES007747-13, R01 ES07747

    Biochemistry 2008;47;48;12886-99

  • Protein kinase D1 mediates NF-kappaB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells.

    Yuan J, Lugea A, Zheng L, Gukovsky I, Edderkaoui M, Rozengurt E and Pandol SJ

    Veterans Affairs Greater Los Angeles Healthcare System, West Los Angeles VA Healthcare Center, Los Angeles, CA 90073, USA. jzyuan@ucla.edu

    The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

    Funded by: NIAAA NIH HHS: P50 AA11999; NIDDK NIH HHS: DK 55003

    American journal of physiology. Gastrointestinal and liver physiology 2008;295;6;G1190-201

  • Hypoxia activates NADPH oxidase to increase [ROS]i and [Ca2+]i through the mitochondrial ROS-PKCepsilon signaling axis in pulmonary artery smooth muscle cells.

    Rathore R, Zheng YM, Niu CF, Liu QH, Korde A, Ho YS and Wang YX

    Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208, USA.

    The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22(phox), p47(phox), and p67(phox) were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47(phox) protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47(phox) gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca(2+) ([ROS](i) and [Ca(2+)](i)), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCepsilon translocation peptide inhibitor, and PKCepsilon gene deletion, but not the conventional PKC inhibitor GO6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and [ROS](i) in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H(2)O(2) removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H(2)O(2) increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS](i) through the mitochondrial ROS-PKCepsilon signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in [ROS](i) and [Ca(2+)](i) as well as contraction in PASMCs.

    Funded by: NHLBI NIH HHS: R01 HL064043, R01 HL064043-07, R01 HL075190, R01 HL075190-04, R01HL064043, R01HL075190; NIEHS NIH HHS: P30 ES006639, P30ES06639

    Free radical biology & medicine 2008;45;9;1223-31

  • Isoform-specific contribution of protein kinase C to prion processing.

    Alfa Cissé M, Louis K, Braun U, Mari B, Leitges M, Slack BE, Fisher A, Auberger P, Checler F and Vincent B

    Institut de Pharmacologie Moléculaire et Cellulaire du CNRS, UMR6097, UNSA, Equipe labellisée Fondation pour la Recherche Médicale, Sophia-Antipolis, Valbonne, France.

    The cellular prion protein (PrP(c)) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrP(c). First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCalpha lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCalpha-, PKCdelta- and PKCepsilon-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCepsilon-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCalpha, delta and epsilon, but not zeta, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production.

    Molecular and cellular neurosciences 2008;39;3;400-10

  • Loss of protein kinase Cepsilon results in impaired cutaneous wound closure and myofibroblast function.

    Leask A, Shi-Wen X, Khan K, Chen Y, Holmes A, Eastwood M, Denton CP, Black CM and Abraham DJ

    CIHR Group in Skeletal Development and Remodeling, Division of Oral Biology and Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London ON, Canada N6A 5C1. andrew.leask@schulich.uwo.ca

    Cutaneous wound repair requires the de novo induction of a specialized form of fibroblast, the alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblast, which migrates into the wound where it adheres to and contracts extracellular matrix (ECM), resulting in wound closure. Persistence of the myofibroblast results in scarring and fibrotic disease. In this report, we show that, compared with wild-type littermates, PKCepsilon-/- mice display delayed impaired cutaneous wound closure and a reduction in myofibroblasts. Moreover, both in the presence and absence of TGFbeta, dermal fibroblasts from PKCepsilon-/- mice cultured on fibronectin show impaired abilities to form ;supermature' focal adhesions and alpha-SMA stress fibers, and reduced pro-fibrotic gene expression. Smad3 phosphorylation in response to TGFbeta1 was impaired in PKCepsilon-/- fibroblasts. PKCepsilon-/- fibroblasts show reduced FAK and Rac activation, and adhesive, contractile and migratory abilities. Overexpressing constitutively active Rac1 rescues the defective FAK phosphorylation, cell migration, adhesion and stress fiber formation of these PKCepsilon-/- fibroblasts, indicating that Rac1 operates downstream of PKCepsilon, yet upstream of FAK. These results suggest that loss of PKCepsilon severely impairs myofibroblast formation and function, and that targeting PKCepsilon may be beneficial in selectively modulating wound healing and fibrotic responses in vivo.

    Journal of cell science 2008;121;Pt 20;3459-67

  • Reactive oxygen species derived from NOX1/NADPH oxidase enhance inflammatory pain.

    Ibi M, Matsuno K, Shiba D, Katsuyama M, Iwata K, Kakehi T, Nakagawa T, Sango K, Shirai Y, Yokoyama T, Kaneko S, Saito N and Yabe-Nishimura C

    Department of Pharmacology and Anatomy and Developmental Biology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

    The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxide-generating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1(-/Y)), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed. In dorsal root ganglia (DRG) neurons of Nox1(+/Y), pretreatment with chemical mediators bradykinin, serotonin, or phorbol 12-myristate 13-acetate (PMA) augmented the capsaicin-induced calcium increase, whereas this increase was significantly attenuated in DRG neurons of Nox1(-/Y). Concomitantly, PMA-induced translocation of PKCepsilon was markedly perturbed in Nox1(-/Y) or Nox1(+/Y) DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCepsilon, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCepsilon but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCepsilon in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;38;9486-94

  • PKCepsilon stimulated arginine methylation of RIP140 for its nuclear-cytoplasmic export in adipocyte differentiation.

    Gupta P, Ho PC, Huq MD, Khan AA, Tsai NP and Wei LN

    Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

    Background: Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that plays roles in diverse metabolic processes including fat accumulation in adipocytes. Previously we identified three methylated arginine residues in RIP140, which rendered its export to the cytoplasm; but it was unclear what triggered RIP140 arginine methylation.

    In this study, we determined the activated PKCepsilon as the specific trigger for RIP140 arginine methylation and its subsequent export. We identified two PKCepsilon-phosphorylated residues of RIP140, Ser-102 and Ser-1003, which synergistically stimulated direct binding of RIP140 by 14-3-3 that recruited protein arginine methyl transferase 1 to methylate RIP140. The methylated RIP140 then preferentially recruited exportin 1 for nuclear export. As a result, the nuclear gene-repressive activity of RIP140 was reduced. In RIP140 null adipocyte cultures, the defect in fat accumulation was effectively rescued by the phosphorylation-deficient mutant RIP140 that resided predominantly in the nucleus, but less so by the phospho-mimetic RIP140 that was exported to the cytoplasm.

    This study uncovers a novel means, via a cascade of protein modifications, to inactivate, or suppress, the nuclear action of an important transcription coregulator RIP140, and delineates the first specific phosphorylation-arginine methylation cascade that could alter protein subcellular distribution and biological activity.

    Funded by: NIDA NIH HHS: DA11190, K02 DA013926, K02-DA13926, R01 DA011190; NIDDK NIH HHS: DK54733, DK60521, R01 DK054733, R01 DK060521

    PloS one 2008;3;7;e2658

  • The scaffold MyD88 acts to couple protein kinase Cepsilon to Toll-like receptors.

    Faisal A, Saurin A, Gregory B, Foxwell B and Parker PJ

    Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, London WC2A 3PX, United Kingdom.

    Mice lacking protein kinase Cepsilon (PKCepsilon) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCepsilon coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCepsilon involvement in TLR signaling and found that PKCepsilon is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKCepsilon binding to 14-3-3beta. LPS-induced PKCepsilon phosphorylation, 14-3-3beta binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKCepsilon phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKCepsilon association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCepsilon. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCepsilon was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKCepsilon at these two sites is required for TLR4- and TLR2-induced NFkappaB reporter activation and IkappaB degradation in reconstituted PKCepsilon(-/-) cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCepsilon recruitment, phosphorylation, and downstream signaling.

    The Journal of biological chemistry 2008;283;27;18591-600

  • Protein kinase C epsilon confers resistance of MCF-7 cells to TRAIL by Akt-dependent activation of Hdm2 and downregulation of p53.

    Shankar E, Sivaprasad U and Basu A

    Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

    Protein kinase C epsilon (PKC epsilon ) acts as an antiapoptotic protein and inhibits tumor necrosis factor-alpha (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in MCF-7 breast cancer cells. Members of the TNF receptor superfamily trigger apoptosis independent of the tumor suppressor protein p53, which primarily affects DNA damage-induced apoptosis. We have previously shown that PKC epsilon acts upstream of Akt to inhibit receptor-initiated cell death. Since Akt can regulate p53, we have examined the involvement of p53 in PKC epsilon-mediated TRAIL resistance. Overexpression of PKC epsilon in MCF-7 cells (MCF-7/PKC epsilon ) caused a decrease in p53 and an increase in human homolog of murine double minute 2 (Hdm2) and phospho-Hdm2. Depletion of p53 by siRNA attenuated, whereas depletion of Hdm2 enhanced TRAIL-mediated apoptosis. Knockdown of Akt decreased Hdm2 phosphorylation, increased p53 level and potentiated TRAIL-induced cell death. Depletion of epsilon from MCF-7 cells caused an increase in p53, whereas knockdown of p53 caused a decrease in Bid mRNA. Depletion of Akt from MCF-7/PKC epsilon cells resulted in an increase in p53 and Bid. These results suggest that PKC epsilon mediates TRAIL resistance by Akt-mediated phosphorylation of Hdm2 resulting in suppression of p53 expression and downregulation of Bid in MCF-7 breast cancer cells.

    Funded by: NCI NIH HHS: CA71727

    Oncogene 2008;27;28;3957-66

  • Protein kinase C epsilon mediation of CRF- and ethanol-induced GABA release in central amygdala.

    Bajo M, Cruz MT, Siggins GR, Messing R and Roberto M

    Molecular and Integrative Neurosciences Department, Committee on Neurobiology of Addictive Disorders, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    In the central amygdala (CeA), ethanol acts via corticotrophin-releasing factor (CRF) type 1 receptors to enhance GABA release. Amygdala CRF mediates anxiety associated with stress and drug dependence, and it regulates ethanol intake. Because mutant mice that lack PKCepsilon exhibit reduced anxiety-like behavior and alcohol consumption, we investigated whether PKCepsilon lies downstream of CRF(1) receptors in the CeA. Compared with PKCepsilon(+/+) CeA neurons, PKCepsilon(-/-) neurons showed increased GABAergic tone due to enhanced GABA release. CRF and ethanol stimulated GABA release in the PKCepsilon(+/+) CeA, but not in the PKCepsilon(-/-) CeA. A PKCepsilon-specific inhibitor blocked both CRF- and ethanol-induced GABA release in the PKCepsilon(+/+) CeA, confirming findings in the PKCepsilon(-/-) CeA. These results identify a PKCepsilon signaling pathway in the CeA that is activated by CRF(1) receptor stimulation, mediates GABA release at nerve terminals, and regulates anxiety and alcohol consumption.

    Funded by: NIAAA NIH HHS: AA013517, AA013588, AA015566, AA06420, AA10994, P50 AA006420, R01 AA013588, R01 AA015566, R37 AA013588, U01 AA013517, U24 AA013517; NIDA NIH HHS: DA03665, R01 DA003665

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;24;8410-5

  • Acrolein consumption exacerbates myocardial ischemic injury and blocks nitric oxide-induced PKCepsilon signaling and cardioprotection.

    Wang GW, Guo Y, Vondriska TM, Zhang J, Zhang S, Tsai LL, Zong NC, Bolli R, Bhatnagar A and Prabhu SD

    Department of Physiology and the Cardiovascular Research Laboratories, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.

    Aldehydes are common reactive constituents of food, water and air. Several food aldehydes are potentially carcinogenic and toxic; however, the direct effects of dietary aldehydes on cardiac ischemia-reperfusion (IR) injury are unknown. We tested the hypothesis that dietary consumption of aldehydes modulates myocardial IR injury and preconditioning. Mice were gavage-fed the alpha, beta-unsaturated aldehyde acrolein (5mg/kg) or water (vehicle) 24h prior to a 30-min coronary artery occlusion and 24-hour reperfusion. Myocardial infarct size was significantly increased in acrolein-treated mice, demonstrating that acute acrolein exposure worsens cardiac IR injury. Furthermore, late cardioprotection afforded by the nitric oxide (NO) donor diethylenetriamine/NO (DETA/NO; dose: 0.1mg/kg x 4, i.v.) was abrogated by the administration of acrolein 2h prior to DETA/NO treatment, indicating that oral acrolein impairs NO donor-induced late preconditioning. To examine potential intracellular targets of aldehydes, we investigated the impact of acrolein on mitochondrial PKCepsilon signaling in the heart. Acrolein-protein adducts were formed in a dose-dependent manner in isolated cardiac mitochondria in vitro and specific acrolein-PKCepsilon adducts were present in cardiac mitochondrial fractions following acrolein exposure in vivo, demonstrating that mitochondria are major targets of aldehyde toxicity. Furthermore, DETA/NO preconditioning induced both PKCepsilon translocation and increased mitochondrial PKCepsilon localization. Both of these responses were blocked by acrolein pretreatment, providing evidence that aldehydes disrupt cardioprotective signaling events involving PKCepsilon. Consumption of an aldehyde-rich diet could exacerbate cardiac IR injury and block NO donor-induced cardioprotection via mechanisms that disrupt PKCepsilon signaling.

    Funded by: NHLBI NIH HHS: HL-43151, HL-55757, HL-78825; NIEHS NIH HHS: ES-11860, ES-12062

    Journal of molecular and cellular cardiology 2008;44;6;1016-22

  • The identification and characterization of novel PKCepsilon phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily.

    Durgan J, Cameron AJ, Saurin AT, Hanrahan S, Totty N, Messing RO and Parker PJ

    Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

    PKCepsilon (protein kinase Cepsilon) is a phospholipid-dependent serine/threonine kinase that has been implicated in a broad array of cellular processes, including proliferation, survival, migration, invasion and transformation. Here we demonstrate that, in vitro, PKCepsilon undergoes autophosphorylation at three novel sites, Ser(234), Ser(316) and Ser(368), each of which is unique to this PKC isoform and is evolutionarily conserved. We show that these sites are phosphorylated over a range of mammalian cell lines in response to a number of different stimuli. Unexpectedly, we find that, in a cellular context, these phosphorylation events can be mediated in-trans by cPKC (classical PKC) isoforms. The functional significance of this cross-talk is illustrated through the observation that the cPKC-mediated phosphorylation of PKCepsilon at residue Ser(368) controls an established PKCepsilon scaffold interaction. Thus our current findings identify three new phosphorylation sites that contribute to the isoform-specific function of PKCepsilon and highlight a novel and direct means of cross-talk between different members of the PKC superfamily.

    Funded by: Medical Research Council; NIAAA NIH HHS: AA013588

    The Biochemical journal 2008;411;2;319-31

  • Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior.

    Lesscher HM, McMahon T, Lasek AW, Chou WH, Connolly J, Kharazia V and Messing RO

    Department of Neurology, Ernest Gallo Clinic and Research Center, University of California at San Francisco, Emeryville, CA 94608, USA.

    Corticotropin-releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCepsilon) null mutant mice, which show reduced anxiety-like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCepsilon activator, psiepsilonRACK, increased levels of pro-CRF, whereas reducing PKCepsilon levels through RNA interference blocked phorbol ester-stimulated increases in CRF. Local knockdown of amygdala PKCepsilon by RNA interference reduced anxiety-like behavior in wild-type mice. Furthermore, local infusion of CRF into the amygdala of PKCepsilon(-/-) mice increased their anxiety-like behavior. These results are consistent with a novel mechanism of PKCepsilon control over anxiety-like behavior through regulation of CRF in the amygdala.

    Funded by: NIAAA NIH HHS: AA013588

    Genes, brain, and behavior 2008;7;3;323-33

  • Role of protein kinase C in selective inhibition of mouse retinal neurites during contacts with chondroitin sulfates.

    Lam JS, Wang L, Lin L and Chan SO

    Department of Anatomy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.

    Chondroitin sulfate proteoglycans elicit a selective inhibition to neurite growth from ventrotemporal (VT) but not dorsonasal (DN) retina, potentiating the bilateral routing of axons in the mouse optic chiasm. We examined whether this selective response is mediated by a difference in protein kinase C (PKC) expression. Effects of suppressing PKC activity in explant preparations of embryonic day 14 retinae with inhibitor Gö6976 or Ro-32-0432 abolished the chondroitin sulfate inhibition to the VT neurites but had no effect to the DN neurites. Whether these responses rely on a difference in expression of PKC in the growth cones was examined using antibodies against six isozymes of PKC. Among these the alpha, betaI and epsilon isozymes were expressed prominently in the retinal growth cones; whilst the betaII, delta and gamma isozymes were barely detected. Moreover, while the alpha and epsilon isozymes were abundant in the filopodial and lamellipodial processes, the betaI isozyme was restricted largely in the core region of the growth cones. Despite these subtype specific localization, there was no significant difference in expression of any of these PKC isozymes between growth cones from VT and DN retina, indicating that the selective response to chondroitin sulfates is not likely generated by a regulation of PKC expression, but by expression of surface molecules that interact with chondroitin sulfate proteoglycans.

    Neuroscience letters 2008;434;1;150-4

  • Islet cell survival during isolation improved through protein kinase C epsilon activation.

    Kvezereli M, Vallentin A, Mochly-Rosen D, Busque S and Fontaine MJ

    Department of Pathology, Stanford University, Stanford, California, USA.

    Strategies inhibiting cell death signaling pathways may enhance the availability of islet transplantation for patients with type 1 diabetes mellitus. The epsilon isoform of protein kinase C (PKC epsilon) has been shown to have an anti-apoptotic effect in many cell types. The present study investigated whether activation of PKC epsilon may improve the yield of functional islet cells for transplantation. Islet cells were isolated from wild-type BALB/c mice preconditioned with either a PKC epsilon activator (psi epsilon RACK) or a TAT carrier control peptide and further treated with the same agents during isolation and in vitro for either 0, 1, 16, or 40 hours. Islet cells were assessed at each time point for viability, apoptosis, and function. psi epsilon RACK-treated islets showed significantly decreased islet cell death up to 40 hours after isolation compared with TAT-treated control islets. Beta-cell function in response to high glucose challenge remained unchanged.

    Transplantation proceedings 2008;40;2;375-8

  • Endothelin-1 induction of Glut1 transcription in 3T3-L1 adipocytes involves distinct PKCepsilon- and p42/p44 MAPK-dependent pathways.

    Kao YS and Fong JC

    Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China.

    We have shown previously that chronic exposure to endothelin-1 (ET-1) may stimulate GLUT1-mediated glucose transport in 3T3-L1 adipocytes via both protein kinase C (PKC)- and mitogen-activated protein kinase (p42/p44 MAPK)-dependent pathways. In the present study, by using a luciferase reporter driven by Glut1 promoter and enhancers (pLuc-GT1/E1/E2) and various constitutively active and dominant negative mutants of PKC isoforms, we identified PKCepsilon as the PKC isoform involved. In addition, we provide evidence that there is no direct interaction between ET-1 activated PKCepsilon and MAPK, at least at the kinase activity level. Furthermore, investigations employing deletion mutants of pLuc-GT1/E1/E2 to locate the putative ET-1 responsive sites and inhibitory agents to suppress the activities of putative transcription factors suggested that transcription factors CREB, Sp1 and NF-kappaB were involved. In summary, the results of this study indicate that ET-1 induction of Glut1 transcription involves distinct PKCepsilon- and MAPK-dependent pathways, as well as downstream transcription factors CREB, Sp1 and NF-kappaB.

    Biochimica et biophysica acta 2008;1780;2;154-9

  • Protein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia.

    Barnett M, Lin D, Akoyev V, Willard L and Takemoto D

    Department of Biochemistry, Chalmers Hall, Kansas State University, Manhattan, KS 66506, USA.

    Protein kinase C (PKC) isoforms have been identified as major cellular signaling proteins that act directly in response to oxidation conditions. In retina and lens two isoforms of PKC respond to changes in oxidative stress, PKCgamma and PKCepsilon, while only PKCepsilon is found in heart. In heart the PKCepsilon acts on connexin 43 to protect from hypoxia. The presence of both isoforms in the lens led to this study to determine if lens PKCepsilon had unique targets. Both lens epithelial cells in culture and whole mouse lens were examined using PKC isoform-specific enzyme activity assays, co-immunoprecipitation, confocal microscopy, immunoblots, and light and electron microscopy. PKCepsilon was found in lens epithelium and cortex but not in the nucleus of mouse lens. The PKCepsilon isoform was activated in both epithelium and whole lens by 5% oxygen when compared to activity at 21% oxygen. In hypoxic conditions (5% oxygen) the PKCepsilon co-immunoprecipitated with the mitochondrial cytochrome c oxidase IV subunit (CytCOx). Concomitant with this the CytCOx enzyme activity was elevated and increased co-localization of CytCOx with PCKvarepsilon was observed using immunolabeling and confocal microscopy. In contrast, no hypoxia-induced activation of CytCOx was observed in lenses from the PKCepsilon knockout mice. Lens from 6-week-old PKCepsilon knockout mice had a disorganized bow region which was filled with vacuoles indicating a possible loss of mitochondria but the size of the lens was not altered. Electron microscopy demonstrated that the nuclei of the PCKepsilon knockout mice were abnormal in shape. Thus, PKCepsilon is found to be activated by hypoxia and this results in the activation of the mitochondrial protein CytCOx. This could protect the lens from mitochondrial damage under the naturally hypoxic conditions observed in this tissue. Lens oxygen levels must remain low. Elevation of oxygen which occurs during vitreal detachment or liquification is associated with cataracts. We hypothesize that elevated oxygen could cause inhibition of PKCepsilon resulting in a loss of mitochondrial protection.

    Funded by: NEI NIH HHS: R01 EY 13421, R01 EY013421, R01 EY013421-07

    Experimental eye research 2008;86;2;226-34

  • Differential roles of the PKC novel isoforms, PKCdelta and PKCepsilon, in mouse and human platelets.

    Pears CJ, Thornber K, Auger JM, Hughes CE, Grygielska B, Protty MB, Pearce AC and Watson SP

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom. catherine.pears@bioch.ox.ac.uk

    Background: Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.

    In this study, we focus on the role of two novel PKC isoforms, PKCdelta and PKCepsilon, in both mouse and human platelets. PKCdelta is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCdelta undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCepsilon, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCepsilon in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRgamma-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.

    Conclusions: These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms delta and epsilon in human and mouse platelets and a selective role for PKCepsilon in signalling through GPVI.

    Funded by: British Heart Foundation; Wellcome Trust

    PloS one 2008;3;11;e3793

  • Identification of transcripts with enriched expression in the developing and adult pancreas.

    Hoffman BG, Zavaglia B, Witzsche J, Ruiz de Algara T, Beach M, Hoodless PA, Jones SJ, Marra MA and Helgason CD

    Department of Cancer Endocrinology, BC Cancer Research Center, West 10th Ave, Vancouver, BC V5Z 1L3, Canada. bhoffman@bccrc.ca

    Background: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.

    Results: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.

    Conclusion: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

    Genome biology 2008;9;6;R99

  • Milk fat globule-EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium.

    Bu HF, Zuo XL, Wang X, Ensslin MA, Koti V, Hsueh W, Raymond AS, Shur BD and Tan XD

    Molecular and Cellular Pathobiology Program and Center for Digestive Disease and Immunobiology, Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60614, USA.

    Milk fat globule-EGF factor 8 (MFG-E8)/lactadherin participates in several cell surface-mediated regulatory events. Although its mRNA is present in the gut, the physiological roles of MFG-E8 in the intestinal mucosa have not been explored. Here we show that MFG-E8 was expressed in intestinal lamina propria macrophages from mice. Using a wound-healing assay, MFG-E8 was shown to promote the migration of intestinal epithelial cells through a PKCepsilon-dependent mechanism. MFG-E8 bound to phosphatidylserine and triggered reorientation of the actin cytoskeleton in intestinal epithelial cells at the wound edge. Depleting MFG-E8 in mice by administration of anti-MFG-E8 antibody or targeted deletion of the MFG-E8 gene resulted in a slowing of enterocyte migration along the crypt-villus axis and focal mucosal injury. Moreover, in septic mice, intestinal MFG-E8 expression was downregulated, which correlated with intestinal injury, interrupted enterocyte migration, and impaired restitution. Treatment with recombinant MFG-E8 restored enterocyte migration, whereas deletion of MFG-E8 impeded mucosal healing in mice with sepsis. These results suggest that a decrease in intestinal MFG-E8 impairs intestinal mucosal repair in sepsis. Together, our data indicate that MFG-E8 plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing and suggest that recombinant MFG-E8 may be beneficial for the treatment of bowel injuries.

    Funded by: NICHD NIH HHS: R01HD23479; NIDDK NIH HHS: R01 DK064240-04, R01DK064240

    The Journal of clinical investigation 2007;117;12;3673-83

  • Protein kinase C epsilon regulates gamma-aminobutyrate type A receptor sensitivity to ethanol and benzodiazepines through phosphorylation of gamma2 subunits.

    Qi ZH, Song M, Wallace MJ, Wang D, Newton PM, McMahon T, Chou WH, Zhang C, Shokat KM and Messing RO

    Ernest Gallo Clinic and Research Center, Department of Neurology, University of California-San Francisco, 5858 Horton Street, Emeryville, CA 94608, USA.

    Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

    Funded by: NIAAA NIH HHS: AA 013588; NIAID NIH HHS: AI/CA 44009

    The Journal of biological chemistry 2007;282;45;33052-63

  • PKCepsilon upregulates voltage-dependent calcium channels in cultured astrocytes.

    Burgos M, Pastor MD, González JC, Martinez-Galan JR, Vaquero CF, Fradejas N, Benavides A, Hernández-Guijo JM, Tranque P and Calvo S

    Unidad de Fisiología, Facultad de Medicina y Centro Regional de Investigaciones Biomedicas, Universidad de Castilla La Mancha, Albacete, Spain.

    Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.

    Glia 2007;55;14;1437-48

  • nPKCepsilon, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells.

    Ehre C, Zhu Y, Abdullah LH, Olsen J, Nakayama KI, Nakayama K, Messing RO and Davis CW

    CCystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248, USA.

    Airway goblet cell mucin secretion is controlled by agonist activation of P2Y(2) purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP(3)), diacylglycerol, Ca(2+) and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKCalpha, nPKCdelta, nPKCepsilon, and nPKCeta; of these, only nPKCdelta translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKCalpha, nPKCdelta, and nPKCeta had the same levels of ATPgammaS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKCepsilon (14.6 and 23.5%, for ATPgammaS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKCepsilon exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPgammaS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y(2)-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKCdelta knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKCepsilon KO mice relative to its WT littermates. We conclude that nPKCepsilon is the effector isoform downstream of P2Y(2)-R activation in the goblet cell secretory response. The translocation of nPKCdelta observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology.

    Funded by: NHLBI NIH HHS: HL-063756

    American journal of physiology. Cell physiology 2007;293;5;C1445-54

  • Inhibition of PKCepsilon improves glucose-stimulated insulin secretion and reduces insulin clearance.

    Schmitz-Peiffer C, Laybutt DR, Burchfield JG, Gurisik E, Narasimhan S, Mitchell CJ, Pedersen DJ, Braun U, Cooney GJ, Leitges M and Biden TJ

    Garvan Institute of Medical Research, Sydney, NSW 2010, Australia. c.schmitz-peiffer@garvan.org.au

    In type 2 diabetes, pancreatic beta cells fail to secrete sufficient insulin to overcome peripheral insulin resistance. Intracellular lipid accumulation contributes to beta cell failure through poorly defined mechanisms. Here we report a role for the lipid-regulated protein kinase C isoform PKCepsilon in beta cell dysfunction. Deletion of PKCepsilon augmented insulin secretion and prevented glucose intolerance in fat-fed mice. Importantly, a PKCepsilon-inhibitory peptide improved insulin availability and glucose tolerance in db/db mice with preexisting diabetes. Functional ablation of PKCepsilon selectively enhanced insulin release ex vivo from diabetic or lipid-pretreated islets and optimized the glucose-regulated lipid partitioning that amplifies the secretory response. Independently, PKCepsilon deletion also augmented insulin availability by reducing both whole-body insulin clearance and insulin uptake by hepatocytes. Our findings implicate PKCepsilon in the etiology of beta cell dysfunction and highlight that enhancement of insulin availability, through separate effects on liver and beta cells, provides a rationale for inhibiting PKCepsilon to treat type 2 diabetes.

    Cell metabolism 2007;6;4;320-8

  • Pharmacological inhibition of epsilon PKC suppresses chronic inflammation in murine cardiac transplantation model.

    Koyanagi T, Noguchi K, Ootani A, Inagaki K, Robbins RC and Mochly-Rosen D

    Department of Chemical and Systems Biology, Stanford University School of Medicine CCSR, Rm 3145A, 269 Campus Drive Stanford, CA 94305-5174, USA.

    Epsilon protein kinase C (epsilonPKC) plays pivotal roles in myocardial infarction and in heart failure. Although cardiac transplantation is a well-established therapy for severe heart failure, allograft rejection and host inflammatory responses limit graft function and reduce life expectancy. Here we determined whether sustained epsilonPKC inhibition beginning 3 days after transplantation suppress allograft rejection and improve cardiac transplantation using a murine heterotopic transplantation model. Hearts of FVB mice (H-2(q)) were transplanted into C57BL/6 mice (H-2(b)). Delivery of the epsilonPKC inhibitor, TAT(47-57)-epsilonV1-2 (epsilonV1-2, n=9, 20 mg/kg/day), or the carrier control peptide, TAT(47-57) (TAT, n=8), by osmotic pump began 3 days after transplantation and continued for the remaining 4 weeks. epsilonV1-2 treatment significantly improved the beating score throughout the treatment. Infiltration of macrophages and T cells into the cardiac grafts was significantly reduced and parenchymal fibrosis was decreased in animals treated with epsilonV1-2 as compared with control treatment. Finally, the rise in pro-fibrotic cytokine, TGF-beta and monocyte recruiting chemokine MCP-1 levels was almost abolished by epsilonV1-2 treatment, whereas the rise in PDGF-BB level was unaffected. These data suggest that epsilonPKC activity contributes to the chronic immune response in cardiac allograft and that an epsilonPKC-selective inhibitor, such as epsilonV1-2, could augment current therapeutic strategies to suppress inflammation and prolong graft survival in humans.

    Funded by: NHLBI NIH HHS: HL52141

    Journal of molecular and cellular cardiology 2007;43;4;517-22

  • Phosphorylation at Ser729 specifies a Golgi localisation for protein kinase C epsilon (PKCepsilon) in 3T3 fibroblasts.

    Xu TR, He G, Dobson K, England K and Rumsby M

    SHWFGF-Proteomics Section, Joseph Black Building, University of Glasgow, University Avenue, Glasgow, G12 8QQ, UK.

    We demonstrate that GFP-PKCepsilon concentrates at a perinuclear site in living fibroblasts and that cell passage induces rapid translocation of PKCepsilon to the periphery where it appears to colocalise with F-actin. When newly passaged cells have adhered and are proliferating again, GFP-PKCepsilon returns to its perinuclear site. GFP-PKCepsilon co-localises with wheat germ agglutinin suggesting that it is associated with the Golgi at the perinuclear site. In support, PKCepsilon is detected in a Golgi-enriched fraction in pre-passage cells but is lost from the fraction after passage. PKCepsilon at the perinuclear Golgi site is phosphorylated at Ser729 but cell passage induces the loss of the phosphate at this site as reported previously [England et al. (2001) J. Biol. Chem. 276, 10437-10442]. PKCepsilon S729A, S729E and S729T mutants, which are not recognised by a specific antiphosphoPKCepsilon (Ser729) antibody, do not concentrate at a perinuclear/Golgi site in proliferating fibroblasts. This suggests that both phosphorylation and serine rather than threonine are needed at position 729 to locate PKCepsilon at its perinuclear/Golgi site. Phorbol ester induced translocation of PKCepsilon to the nucleus also requires dephosphorylation at Ser729; after translocation nuclear PKCepsilon lacks a phosphate at Ser729. Sulphation and secretion of glycosaminoglycan (GAG) chains from fibroblasts increases on passage and returns to basal as cells proliferate showing that cell passage influences secretory events at the Golgi. The results indicate that Ser729 phosphorylation plays a role in determining PKCepsilon localisation in fibroblasts.

    Cellular signalling 2007;19;9;1986-95

  • Protein kinase Cepsilon (PKCepsilon) and Src control PKCdelta activation loop phosphorylation in cardiomyocytes.

    Rybin VO, Guo J, Gertsberg Z, Elouardighi H and Steinberg SF

    Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.

    Protein kinase Cdelta (PKCdelta) is unusual among AGC kinases in that it does not require activation loop (Thr(505)) phosphorylation for catalytic competence. Nevertheless, Thr(505) phosphorylation has been implicated as a mechanism that influences PKCdelta activity. This study examines the controls of PKCdelta-Thr(505) phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCdelta autophosphorylation in the "priming" maturational PKCdelta-Thr(505) phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCdelta-Thr(505) phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the alpha(1)-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCepsilon overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCdelta-Thr(505) autophosphorylation and PKCdelta-Thr(505) phosphorylation in trans by PKCepsilon. Our studies show that adenovirus-mediated PKCepsilon overexpression leads to an increase in PKCdelta-Thr(505) phosphorylation. However, this cannot be attributed to an effect of PKCepsilon to function as a direct PKCdelta-Thr(505) kinase, since the PKCepsilon-dependent increase in PKCdelta-Thr(505) phosphorylation is accompanied by (and dependent upon) increased PKCdelta phosphorylation at Tyr(311) and Tyr(332). Further studies implicate Src in this mechanism, showing that 1) PKCepsilon overexpression increases PKCdelta-Thr(505) phosphorylation in cardiomyocytes and Src(+) cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCdelta-Tyr(311)/Tyr(332) phosphorylation), and 2) in vitro PKCdelta-Thr(505) autophosphorylation is augmented in assays performed with Src (which promotes PKCdelta-Tyr(311)/Tyr(332) phosphorylation). Collectively, these results identify a novel PKCdelta-Thr(505) autophosphorylation mechanism that is triggered by PKCepsilon overexpression and involves Src-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation.

    Funded by: NHLBI NIH HHS: HL77860, R01 HL077860, R01 HL077860-04

    The Journal of biological chemistry 2007;282;32;23631-8

  • Fatty acid represses insulin receptor gene expression by impairing HMGA1 through protein kinase Cepsilon.

    Dey D, Bhattacharya A, Roy S and Bhattacharya S

    Molecular Endocrinology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India.

    It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p <0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKCepsilon was also inhibited. Surprisingly, phosphorylation of cytosolic PKCepsilon was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKCepsilon translocation inhibitor peptide, epsilonV1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKCepsilon migration to implement palmitate effect. Experimental evidences indicate that phospho-PKCepsilon adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity.

    Biochemical and biophysical research communications 2007;357;2;474-9

  • Increased response to morphine in mice lacking protein kinase C epsilon.

    Newton PM, Kim JA, McGeehan AJ, Paredes JP, Chu K, Wallace MJ, Roberts AJ, Hodge CW and Messing RO

    Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, CA, USA.

    The protein kinase C (PKC) family of serine-threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCepsilon have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCepsilon null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCepsilon null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of mu-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCepsilon as a key regulator of opiate sensitivity in mice.

    Funded by: NIAAA NIH HHS: AA013588, AA014983, P60 AA011605-100008, R01 AA014983-03

    Genes, brain, and behavior 2007;6;4;329-38

  • Increased sensitivity to the aversive effects of ethanol in PKCepsilon null mice revealed by place conditioning.

    Newton PM and Messing RO

    Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, CA, USA.

    Determining the intracellular signaling pathways that mediate the rewarding effects of ethanol may help identify drug targets to curb excessive alcohol consumption. Mice lacking the epsilon isoform of protein kinase C (PKCepsilon) voluntarily consumed less ethanol than wild-type mice in two-bottle choice and operant self-administration assays. Decreased consumption may reflect either increased or decreased sensitivity to the rewarding effects of ethanol. Alternatively, decreased voluntary consumption may reflect a change in sensitivity to the aversive effects of ethanol. The authors used place conditioning to determine that PKCepsilon null mice have an increased sensitivity to the aversive effects of ethanol but a decreased sensitivity to the rewarding effects of ethanol. Together these data suggest that PKCepsilon null mice voluntarily consume less ethanol because they derive less reward and are more sensitive to the aversive effects of ethanol.

    Behavioral neuroscience 2007;121;2;439-42

  • Peptides derived from the C2 domain of protein kinase C epsilon (epsilon PKC) modulate epsilon PKC activity and identify potential protein-protein interaction surfaces.

    Brandman R, Disatnik MH, Churchill E and Mochly-Rosen D

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

    Peptides derived from protein kinase C (PKC) modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919-924). We previously demonstrated that the C2 domain of PKC plays a critical role in these interactions. By focusing on epsilonPKC and using a rational approach, we then identified one C2-derived peptide that acts as an isozyme-selective activator and another that acts as a selective inhibitor of epsilonPKC. These peptides were used to identify the role of epsilonPKC in protection from cardiac and brain ischemic damage, in prevention of complications from diabetes, in reducing pain, and in protecting transplanted hearts. The efficacy of these two peptides led us to search for additional C2-derived peptides with PKC-modulating activities. Here we report on the activity of a series of 5-9-residue peptides that are derived from regions that span the length of the C2 domain of epsilonPKC. These peptides were tested for their effect on PKC activity in cells in vivo and in an ex vivo model of acute ischemic heart disease. Most of the peptides acted as activators of PKC, and a few peptides acted as inhibitors. PKC-dependent myristoylated alanine-rich C kinase substrate phosphorylation in epsilonPKC knock-out cells revealed that only a subset of the peptides were selective for epsilonPKC over other PKC isozymes. These epsilonPKC-selective peptides were also protective of the myocardium from ischemic injury, an epsilonPKC-dependent function (Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell. Cardiol. 31, 1937-1948), and caused selective translocation of epsilonPKC over other isozymes when injected systemically into mice. Examination of the structure of the C2 domain from epsilonPKC revealed that peptides with similar activities clustered into discrete regions within the domain. We propose that these regions represent surfaces of protein-protein interactions within epsilonPKC and/or between epsilonPKC and other partner proteins; some of these interactions are unique to epsilonPKC, and others are common to other PKC isozymes.

    Funded by: NHLBI NIH HHS: HL52141

    The Journal of biological chemistry 2007;282;6;4113-23

  • Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.

    Munton RP, Tweedie-Cullen R, Livingstone-Zatchej M, Weinandy F, Waidelich M, Longo D, Gehrig P, Potthast F, Rutishauser D, Gerrits B, Panse C, Schlapbach R and Mansuy IM

    Brain Research Institute, Medical Faculty of the University of Zürich, Switzerland.

    Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

    Molecular & cellular proteomics : MCP 2007;6;2;283-93

  • Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells.

    Rathore R, Zheng YM, Li XQ, Wang QS, Liu QH, Ginnan R, Singer HA, Ho YS and Wang YX

    Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208, USA.

    The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.

    Funded by: NHLBI NIH HHS: R01 HL064043, R01 HL075190

    Biochemical and biophysical research communications 2006;351;3;784-90

  • Fucoidan prevents C epsilon germline transcription and NFkappaB p52 translocation for IgE production in B cells.

    Oomizu S, Yanase Y, Suzuki H, Kameyoshi Y and Hide M

    Hiroshima Prefectural Institute of Industrial Science and Technology, Higashi-Hiroshima, Japan.

    Fucoidan, a dietary fiber contained in seaweed, reduces the increase of antigen-specific IgE in mice exposed to ovalbumin. In this study, we investigated the effect of fucoidan on IgE production and intracellular events in B cells in vitro. Fucoidan inhibited the production of IgE and C epsilon germline transcription in murine B cells induced by IL-4 (100 ng/ml) and anti-CD40 antibodies (10 microg/ml), whereas it stimulated cell proliferation. A significant effect of fucoidan on IgE production was observed when B cells were stimulated with a higher dose (5 microg/ml) of anti-CD40 antibodies, but not when stimulated with lower doses (1.25, 2.5 microg/ml), regardless of the IL-4 concentrations. Moreover, nuclear translocation of NFkappaB p52, but neither that of NFkappaB p65, nor the phosphorylation of JAK1 and STAT6 was reduced by fucoidan. These results suggest that fucoidan inhibited IgE production by preventing the NFkappaB p52-mediated pathways activated by CD40.

    Biochemical and biophysical research communications 2006;350;3;501-7

  • Cdc42 is involved in PKCepsilon- and delta-induced neurite outgrowth and stress fibre dismantling.

    Trollér U and Larsson C

    Lund University, Department of Laboratory Medicine, Molecular Medicine, Entrance 78, 3rd floor, Malmö University Hospital, SE-205 02 Malmö, Sweden.

    We have shown that protein kinase C (PKC)epsilon, independently of the catalytic domain, induces outgrowth of cellular processes via its regulatory domain in both neural cells and fibroblasts. This was accompanied by stress fibre loss. Here, we have examined the role of the small GTPases, Rac1, and Cdc42, in these PKC-mediated morphological and cytoskeletal changes. Both constitutively active and dominant negative Rac1 and Cdc42 attenuated the PKC-mediated outgrowth of processes. The suppression was larger for Cdc42 than for Rac1. The PKC-mediated dismantling of the stress fibres in both HiB5 and fibroblasts was inhibited by the expression of the Cdc42 mutants whereas they had smaller effects on the stress fibre dismantling induced by the ROCK inhibitor, Y-27632, indicating a more crucial role for Cdc42 in the PKC-mediated pathway. We conclude that Cdc42 is an important downstream factor in the pathway through which PKC mediates morphological and cytoskeletal effects.

    Biochemical and biophysical research communications 2006;349;1;91-8

  • Protective role of protein kinase C epsilon activation in ischemia-reperfusion arrhythmia.

    Yue Y, Qu Y and Boutjdir M

    VA New York Harbor Healthcare System, SUNY Downstate Medical Center, Brooklyn, NY 11209 and NYU School of Medicine, NY 10010, USA.

    Purpose: Ischemic heart disease carries an increased risk of malignant ventricular tachycardia (VT), fibrillation (VF), and sudden cardiac death. Protein kinase C (PKC) epsilon activation has been shown to improve the hemodynamics in hearts subjected to ischemia/reperfusion. However, very little is known about the role of epsilon PKC in reperfusion arrhythmias. Here we show that epsilon PKC activation is anti-arrhythmic and its inhibition is pro-arrhythmic.

    Method: Langendorff-perfused isolated hearts from epsilonPKC agonist (epsilonPKC activation), antagonist (epsilonPKC inhibition) transgenic (TG), and wild-type control mice were subjected to 30 min stabilization period, 10 min global ischemia, and 30 min reperfusion. Action potentials (APs) and calcium transients (CaiT) were recorded simultaneously at 37 degrees C using optical mapping techniques. The incidence of VT and VF was assessed during reperfusion.

    Results: No VT/VF was seen in any group during the stabilization period in which hearts were perfused with Tyrode's solution. Upon reperfusion, 3 out of the 16 (19%) wild-type mice developed VT but no VF. In epsilonPKC antagonist group, in which epsilonPKC activity was downregulated, 10 out of 13 (76.9%) TG mice developed VT, of which six (46.2%) degenerated into sustained VF upon reperfusion. Interestingly, in epsilonPKC agonist mice, in which the activity of epsilonPKC was upregulated, no VF was observed and only 1 out of 12 mice showed only transient VT during reperfusion. During ischemia and reperfusion, CaiT decay was exceedingly slower in the antagonist mice compared to the other two groups.

    Conclusion: Moderate in vivo activation of epsilonPKC exerts beneficial antiarrhythmic effect vis-a-vis the lethal reperfusion arrhythmias. Abnormal CaiT decay may, in part, contribute to the high incidence of reperfusion arrhythmias in the antagonist mice. These findings have important implications for the development of PKC isozyme targeted therapeutics and subsequently for the treatment of ischemic heart diseases.

    Funded by: NHLBI NIH HHS: R01 HL-077494

    Biochemical and biophysical research communications 2006;349;1;432-8

  • Cardioprotection initiated by reactive oxygen species is dependent on activation of PKCepsilon.

    Kabir AM, Clark JE, Tanno M, Cao X, Hothersall JS, Dashnyam S, Gorog DA, Bellahcene M, Shattock MJ and Marber MS

    Dept. of Cardiology, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK.

    To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on protein kinase Cepsilon (PKCepsilon), isolated buffer-perfused mouse hearts were randomized to four groups: 1) antimycin A (AA) (0.1 microg/ml) for 3 min followed by 10 min washout and then 30 min global ischemia (I) and 2 h reperfusion (R); 2) controls of I/R alone; 3) AA bracketed with 13 min of N-2-mercaptopropionyl- glycine (MPG) followed by I/R; and 4) MPG (200 microM) alone, followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1 microg/ml), and lucigenin was used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1 microg/ml) with or without MPG, and PKCepsilon translocation was measured by cell fractionation and subsequent Western blot analysis. Finally, the dependence of AA protection on PKCepsilon was determined by the use of knockout mice (-/-) lacking PKCepsilon. AA exposure caused ROS production, which was abolished by the mitochondrial uncoupler mesoxalonitrile 4-trifluoromethoxyphenylhydrazone. In addition, AA significantly reduced the percent infarction-left ventricular volume compared with control I/R (26 +/- 4 vs. 43 +/- 2%; P < 0.05). Bracketing AA with MPG caused a loss of protection (52 +/- 7 vs. 26 +/- 4%; P < 0.05). AA caused PKCepsilon translocation only in the absence of MPG, and protection was lost on the pkcepsilon(-/-) background (38 +/- 3 vs. 15 +/- 4%; P < 0.001). AA causes ROS production, on which protection and PKCepsilon translocation depend. In addition, protection is absent in PKCepsilon null hearts. Our results imply that, in common with ischemic preconditioning, PKCepsilon is crucial to ROS-mediated protection.

    Funded by: Medical Research Council: G0001112; Wellcome Trust: 02/105/14432

    American journal of physiology. Heart and circulatory physiology 2006;291;4;H1893-9

  • Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice.

    Amadesi S, Cottrell GS, Divino L, Chapman K, Grady EF, Bautista F, Karanjia R, Barajas-Lopez C, Vanner S, Vergnolle N and Bunnett NW

    Department of Surgery, University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, CA 94143-0660, USA.

    Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

    Funded by: NIDDK NIH HHS: DK39957, DK43207, DK57480

    The Journal of physiology 2006;575;Pt 2;555-71

  • Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon.

    Marín YE, Namkoong J, Cohen-Solal K, Shin SS, Martino JJ, Oka M and Chen S

    Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, 164 Frelinghuysen Rd., Piscataway, NJ 08854, USA.

    Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.

    Funded by: NCI NIH HHS: F31CA103364, R01CA108720; NIEHS NIH HHS: ES05022; NIGMS NIH HHS: GM55145

    Cellular signalling 2006;18;8;1279-86

  • Trif-related adapter molecule is phosphorylated by PKC{epsilon} during Toll-like receptor 4 signaling.

    McGettrick AF, Brint EK, Palsson-McDermott EM, Rowe DC, Golenbock DT, Gay NJ, Fitzgerald KA and O'Neill LA

    School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.

    PKCepsilon has been shown to play a key role in the effect of the Gram-negative bacterial product LPS; however, the target for PKCepsilon in LPS signaling is unknown. LPS signaling is mediated by Toll-like receptor 4, which uses four adapter proteins, MyD88, MyD88 adapter-like (Mal), Toll/IL-1R domain-containing adapter inducing IFN-beta (Trif), and Trif-related adapter molecule (TRAM). Here we show that TRAM is transiently phosphorylated by PKCepsilon on serine-16 in an LPS-dependent manner. Activation of IFN regulatory factor 3 and induction of the chemokine RANTES, which are both TRAM-dependent, were attenuated in PKCepsilon-deficient cells. TRAMS16A is inactive when overexpressed and is attenuated in its ability to reconstitute signaling in TRAM-deficient cells. We have therefore uncovered a key process in Toll-like receptor 4 signaling, identifying TRAM as the target for PKCepsilon.

    Funded by: Medical Research Council: G0400007, G1000133

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;24;9196-201

  • The very C-terminus of protein kinase Cepsilon is critical for the full catalytic competence but its hydrophobic motif is dispensable for the interaction with 3-phosphoinositide-dependent kinase-1.

    Zhu Y, Smith D, Verma C, Lim WG, Tan BJ, Armstrong JS, Zhou S, Chan E, Tan SL, Zhu YZ, Cheung NS and Duan W

    Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Drive, 117597, Singapore.

    In this article, we explore the role of the C-terminus (V5 domain) of PKCepsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCepsilon resulted in a PKCepsilon-Delta731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCepsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCepsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCepsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCepsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.

    Cellular signalling 2006;18;6;807-18

  • PKCepsilon increases endothelin converting enzyme activity and reduces amyloid plaque pathology in transgenic mice.

    Choi DS, Wang D, Yu GQ, Zhu G, Kharazia VN, Paredes JP, Chang WS, Deitchman JK, Mucke L and Messing RO

    Ernest Gallo Clinic and Research Center, Emeryville, CA 94608, USA.

    Deposition of plaques containing amyloid beta (Abeta) peptides is a neuropathological hallmark of Alzheimer's disease (AD). Here we demonstrate that neuronal overexpression of the epsilon isozyme of PKC decreases Abeta levels, plaque burden, and plaque-associated neuritic dystrophy and reactive astrocytosis in transgenic mice expressing familial AD-mutant forms of the human amyloid precursor protein (APP). Compared with APP singly transgenic mice, APP/PKCepsilon doubly transgenic mice had decreased Abeta levels but showed no evidence for altered cleavage of APP. Instead, PKCepsilon overexpression selectively increased the activity of endothelin-converting enzyme, which degrades Abeta. The activities of other Abeta-degrading enzymes, insulin degrading enzyme and neprilysin, were unchanged. These results indicate that increased neuronal PKCepsilon activity can promote Abeta clearance and reduce AD neuropathology through increased endothelin-converting enzyme activity.

    Funded by: NIA NIH HHS: AG022074, AG05834, AG11385, F32 AG005834, P01 AG022074, R01 AG011385, R37 AG011385; NIAAA NIH HHS: AA013588, R01 AA013588, R37 AA013588; NINDS NIH HHS: NS41787, R01 NS041787

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;21;8215-20

  • A redundant role for PKC-epsilon in mast cell signaling and effector function.

    Lessmann E, Leitges M and Huber M

    Department of Molecular Immunology, Institute for Biology III, University of Freiburg and Max Planck Institute for Immunobiology, Stübeweg 51, 79108 Freiburg, Germany.

    Protein kinase (PK) C-epsilon is strongly expressed in mast cells (MCs) and activated in response to antigen-mediated high-affinity receptor for IgE (Fc epsilonR1) engagement. A critical role of PKC-epsilon in antigen-triggered activation of various signaling pathways was observed in basophilic leukemia cells. To study the function of PKC-epsilon in MCs differentiated in vitro from murine bone marrow, we used our established PKC-epsilon null mice. Unexpectedly, we did not reveal any difference in antigen-induced activation of many central signaling molecules (PKB, mitogen-activated protein kinase, p38, Jun-N-terminal kinase, phospholipase C-gamma1, Bruton's tyrosine kinase, PKD, Fos and PKC-delta) in time-course as well as dose-response studies between PKC-epsilon-deficient and wild-type MCs. In correlation, antigen-triggered degranulation, release of arachidonic acid and secretion of IL-6 were unaltered by the loss of PKC-epsilon. Furthermore, stimulation of MCs via different receptor systems [Steel factor receptor (c-kit) and toll-like receptor 4] did not lead to differences in the measured responses between both cell types. These results strongly suggest that PKC-epsilon plays a redundant role in MCs stimulated by antigen as well as other well-known MC stimuli.

    International immunology 2006;18;5;767-73

  • Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects.

    Desai J, Shannon ME, Johnson MD, Ruff DW, Hughes LA, Kerley MK, Carpenter DA, Johnson DK, Rinchik EM and Culiat CT

    Graduate School for Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory, 1060 Commerce Park, Oak Ridge, TN 37831, USA.

    The mammalian Nell1 gene encodes a protein kinase C-beta1 (PKC-beta1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell1(6R), a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell1(6R) has a T-->A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell1(6R) mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell1(6R) mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

    Human molecular genetics 2006;15;8;1329-41

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Involvement of protein kinase Cepsilon in the negative regulation of Akt activation stimulated by granulocyte colony-stimulating factor.

    Liu H, Qiu Y, Xiao L and Dong F

    Department of Biological Sciences, University of Toledo, OH 43606, USA.

    Stimulation of cells with G-CSF activates multiple signaling cascades, including the serine/threonine kinase Akt pathway. We show in this study that G-CSF-induced activation of Akt in myeloid 32D was specifically inhibited by treatment with PMA, a protein kinase C (PKC) activator. PMA treatment also rapidly attenuated sustained Akt activation mediated by a carboxy truncated G-CSF receptor, expressed in patients with acute myeloid leukemia evolving from severe congenital neutropenia. The inhibitory effect of PMA was abolished by pretreatment of cells with specific PKC inhibitor GF109203X, suggesting that the PKC pathway negatively regulates Akt activation. Ro31-8820, a PKCepsilon inhibitor, also abrogated PMA-mediated inhibition of Akt activation, whereas rottlerin and Go6976, inhibitors of PKCdelta and PKCalphabetaI, respectively, exhibited no significant effects. Furthermore, overexpression of the wild-type and a constitutively active, but not a kinase-dead, forms of PKCepsilon markedly attenuated Akt activation, and inhibited the proliferation and survival of cells in response to G-CSF. The expression of PKCepsilon was down-regulated with G-CSF-induced terminal granulocytic differentiation. Together, these results implicate PKCepsilon as a negative regulator of Akt activation stimulated by G-CSF and indicate that PKCepsilon plays a negative role in cell proliferation and survival in response to G-CSF.

    Funded by: NCI NIH HHS: CA88815, R01CA92172

    Journal of immunology (Baltimore, Md. : 1950) 2006;176;4;2407-13

  • Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes.

    Li L, Sampat K, Hu N, Zakari J and Yuspa SH

    Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. lilu@mail.nih.gov

    Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

    The Journal of biological chemistry 2006;281;6;3237-43

  • Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.

    Littler CM, Wehling CA, Wick MJ, Fagan KA, Cool CD, Messing RO and Dempsey EC

    Cardiovascular Pulmonary Research Laboratory, B-133, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Denver, CO 80262, USA. Cassana.Littler@uchsc.edu

    Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.

    Funded by: NHLBI NIH HHS: HL 14985; NIDDK NIH HHS: 5T35 DK 07496-18

    American journal of physiology. Lung cellular and molecular physiology 2005;289;6;L1083-93

  • PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility.

    Ivaska J, Vuoriluoto K, Huovinen T, Izawa I, Inagaki M and Parker PJ

    VTT Technical Research Centre for Finland, Medical Biotechnology and University of Turku Centre for Biotechnology, Turku, Finland.

    PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.

    The EMBO journal 2005;24;22;3834-45

  • Identification of an inactivating cysteine switch in protein kinase Cepsilon, a rational target for the design of protein kinase Cepsilon-inhibitory cancer therapeutics.

    Chu F, Koomen JM, Kobayashi R and O'Brian CA

    Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

    Critical roles played by some protein kinases in neoplastic transformation and progression provide a rationale for developing selective, small-molecule kinase inhibitors as antineoplastic drugs. Protein kinase Cepsilon (PKCepsilon) is a rational target for cancer therapy, because it is oncogenic and prometastatic in transgenic mouse models. PKCepsilon is activated by sn-1,2-diacylglycerol (DAG). Attempts to develop selective PKCepsilon inhibitors that block activation by DAG or compete with ATP have not yet met with success, suggesting a need for new strategies. We previously reported that cystamine and a metabolic cystine precursor inactivate PKCepsilon in cells in a thiol-reversible manner. In this report, we first determined that PKCepsilon became resistant to inactivation by disulfides when Cys452 was replaced with alanine by site-specific mutagenesis of human PKCepsilon or a constitutively active PKCepsilon mutant. These results showed that the disulfides inactivated PKCepsilon by thiol-disulfide exchange, either upon Cys452 S-thiolation or by rearrangement to an intra-protein disulfide. Mass spectrometric analysis of peptide digests of cystamine-inactivated, carbamidomethylated PKCepsilon detected a peptide S-cysteaminylated at Cys452, indicating that Cys452 S-cysteaminylation is a stable modification. Furthermore, PKCepsilon inactivation by N-ethylmaleimide was Cys452 dependent, providing corroborative evidence that PKCepsilon inhibitors can be designed by targeting Cys452 with small molecules that stably modify the residue. Cys452 is an active site residue that is conserved in only 11 human protein kinase genes. Therefore, the PKCepsilon-inactivating Cys452 switch is a rational target for the design of antineoplastic drugs that selectively inhibit PKCepsilon.

    Funded by: NCI NIH HHS: CA16672, R01 CA108534

    Cancer research 2005;65;22;10478-85

  • Protein kinase C epsilon induces systolic cardiac failure marked by exhausted inotropic reserve and intact Frank-Starling mechanism.

    Montgomery DE, Rundell VL, Goldspink PH, Urboniene D, Geenen DL, de Tombe PP and Buttrick PM

    Department of Medicine, Section of Cardiology, Univ. of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA.

    Myofilament dysfunction is a common point of convergence for many forms of heart failure. Recently, we showed that cardiac overexpression of PKC epsilon initially depresses myofilament activity and then leads to a progression of changes characteristic of human heart failure. Here, we examined the effects of PKC epsilon on contractile reserve, Starling mechanism, and myofilament activation in this model of end-stage dilated cardiomyopathy. Pressure-volume loop analysis and echocardiography showed that the PKC epsilon mice have markedly compromised systolic function and increased end-diastolic volumes. Dobutamine challenge resulted in a small increase in contractility in PKC epsilon mice but failed to enhance cardiac output. The PKC epsilon mice showed a normal length-dependent tension development in skinned cardiac muscle preparations, although Frank-Starling mechanism appeared to be compromised in the intact animal. Simultaneous measurement of tension and ATPase demonstrated that the maximum tension and ATPase were markedly lower in the PKC epsilon mice at any length or Ca2+ concentration. However, the tension cost was also lower indicating less energy expenditure. We conclude 1) that prolonged overexpression of PKC epsilon ultimately leads to a dilated cardiomyopathy marked by exhausted contractile reserve, 2) that PKC epsilon does not compromise the Frank-Starling mechanism at the myofilament level, and 3) that the Starling curve excursion is limited by the inotropic state of the heart. These results reflect the significance of the primary myofilament contractilopathy induced by phosphorylation and imply a role for PKC epsilon-mediated phosphorylation in myofilament physiology and the pathophysiology of decompensated cardiac failure.

    Funded by: NHLBI NIH HHS: HL-63704, P01-HL-62426

    American journal of physiology. Heart and circulatory physiology 2005;289;5;H1881-8

  • Suppression of protein kinase Cepsilon mediates 17beta-estradiol-induced neuroprotection in an immortalized hippocampal cell line.

    Jung ME, Watson DG and Simpkins JW

    Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA. mjung@hsc.unt.edu

    Although estrogens are neuroprotective in a variety of neuroprotection models, the precise underlying mechanisms are currently not well understood. Here, we examined the role of protein kinase C (PKC) in mediating estrogen-induced neuroprotection in the HT-22 immortalized hippocampal cell line. The neuroprotection model utilized calcein fluorescence to quantitate cell viability following glutamate insults. 17beta-Estradiol (betaE2) protected HT-22 cells when treatment was initiated before or after the glutamate insult. The inhibition of PKC by bis-indolylmaleimide mimicked and enhanced betaE2-induced neuroprotection. In contrast, the inhibition of specific PKC isozymes (alpha and beta) by Go6976, inhibition of 1-phosphatidylinositol 3 kinase by wortmannin, or inhibition of protein kinase A by H-89, did not alter cell viability, suggesting a specific involvement of PKC in an isozyme-dependent manner. We further examined whether estrogen interacts with PKC in a PKC isozyme-specific manner. Protein levels and activity of PKC isozymes (alpha, delta, epsilon, and zeta) were assessed by western blot analysis and radiolabeled phosphorylation assays respectively. Among the isozymes tested, betaE2 altered only PKCepsilon; it reduced the activity and membrane translocation of PKCepsilon in a manner that correlated with its protection against glutamate toxicity. Furthermore, betaE2 reversed the increased activity of membrane PKCepsilon induced by glutamate. These data suggest that the neuroprotective effects of estrogens are mediated in part by inhibition of PKCepsilon activity and membrane translocation.

    Funded by: NIA NIH HHS: AG10485, AG22550; NIAAA NIH HHS: AA013864

    Journal of neurochemistry 2005;95;3;745-55

  • Expression profile of protein kinase C isozymes in preimplantation mouse development.

    Dehghani H and Hahnel AC

    Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.

    In the preimplantation mouse embryo, the protein kinase C (PKC) family has been implicated in regulation of egg activation, progression of meiotic and mitotic cell cycles, embryo compaction, and blastulation, but the involvement of the individual isozymes is largely unknown. Here, using semiquantitative immunocytochemistry and confocal microscopy we analyze the relative amount and subcellular distribution of ten isozymes of PKC (alpha, betaI, betaII, gamma, delta, epsilon, eta, theta, zeta, iota/lambda) and a PKC-anchoring protein, receptor for activated C-kinase 1 (RACK1). Our results show that all of these isoforms of PKC are present between the two-cell and blastocyst stages of mouse preimplantation development, and that each has a distinct, dynamic pattern and level of expression. The data suggest that different complements of the isozymes are involved in various steps of preimplantation development, and will serve as a framework for further functional studies of the individual isozymes. In particular, there was a transient increase in the nuclear concentration of several isozymes at the early four-cell stage, suggesting that some of the PKC isozymes might be involved in regulation of nuclear organization and function in the early mouse embryo.

    Reproduction (Cambridge, England) 2005;130;4;441-51

  • PKC-{epsilon}-dependent survival signals in diabetic hearts.

    Malhotra A, Begley R, Kang BP, Rana I, Liu J, Yang G, Mochly-Rosen D and Meggs LG

    Division of Nephrology, Department of Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, USA. Malhotas@umdnj.edu

    Diabetes mellitus is complicated by the development of a primary cardiomyopathy, which contributes to the excess morbidity and mortality of this disorder. The protein kinase C (PKC) family of isozymes plays a key role in the cardiac phenotype expressed during postnatal development and in response to pathological stimuli. Hyperglycemia is an activating signal for cardiac PKC isozymes that modulate a myriad of cell events including cell death and survival. The epsilon-isozyme of the PKC family transmits a powerful survival signal in cardiac muscle cells. Accordingly, to test the hypothesis that endogenous activation of cardiac PKC-epsilon will protect against hyperglycemic cell injury and left ventricular dysfunction, diabetes mellitus was induced using streptozotocin in genetically engineered mice with cardiac-specific expression of the PKC-epsilon translocation activator [psiepsilon-receptors for activated C kinase (psiepsilon-RACK)]. The results demonstrate a striking PKC-epsilon cardioprotective phenotype in diabetic psiepsilon-RACK (epsilon-agonist) mice that is characterized by inhibition of the hyperglycemia apoptosis signal, attenuation of hyperglycemia-mediated oxidative stress, and preservation of parameters of left ventricular pump function. Hearts of diabetic epsilon-agonist mice exhibited selective trafficking of PKC-epsilon to membrane and mitochondrial compartments, phosphorylation/inactivation of the mitochondrial Bad protein, and inhibition of cytochrome c release. We conclude that activation of endogenous PKC-epsilon in hearts of diabetic epsilon-agonist mice promotes the survival phenotype, attenuates markers of oxidative stress, and inhibits the negative inotropic properties of chronic hyperglycemia.

    Funded by: NHLBI NIH HHS: HL-59139, HL-69020; NIA NIH HHS: AG-014121

    American journal of physiology. Heart and circulatory physiology 2005;289;4;H1343-50

  • Subcellular localization of protein kinase C delta and epsilon affects transcriptional and post-transcriptional processes in four-cell mouse embryos.

    Dehghani H, Reith C and Hahnel AC

    Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.

    During mouse preimplantation development, two isozymes of protein kinase C (PKC), delta and epsilon, transiently localize to nuclei at the early four-cell stage. In order to study their functions at this stage, we altered the subcellular localization of these isozymes (ratio of nuclear to cytoplasmic concentrations) with peptides that specifically activate or inhibit translocation of each isozyme. The effects of altering nuclear concentration of each isozyme on transcription (5-bromouridine 5'-triphosphate (BrUTP) incorporation), amount and distribution of small nuclear ribonucleoproteins (snRNPs), nucleolar dynamics (immunocytochemistry for Smith antigen (Sm) protein) and the activity of embryonic alkaline phosphatase (EAP; histochemistry) were examined. We found that nuclear concentration of PKC epsilon correlated with total mRNA transcription. Higher nuclear concentrations of both PKC delta and epsilon decreased storage of snRNPs in Cajal bodies and decreased the number of nucleoli, but did not affect the nucleoplasmic concentration of snRNPs. Inhibiting translocation of PKC delta out of the nucleus at the early four-cell stage decreased cytoplasmic EAP activity, whereas inhibiting translocation of PKC epsilon increased EAP activity slightly. These results indicate that translocation of PKC delta and epsilon in and out of nuclei at the early four-cell stage in mice can affect transcription or message processing, and that sequestration of these PKC in nuclei can also affect the activity of a cytoplasmic protein (EAP).

    Reproduction (Cambridge, England) 2005;130;4;453-65

  • Protein kinase C epsilon mediates angiotensin II-induced activation of beta1-integrins in cardiac fibroblasts.

    Stawowy P, Margeta C, Blaschke F, Lindschau C, Spencer-Hänsch C, Leitges M, Biagini G, Fleck E and Graf K

    Department of Medicine-Cardiology, Deutsches Herzzentrum Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.

    Objective: Angiotensin II (AII) promotes cardiac fibrosis by increased extracellular matrix production and enhanced interaction of matrix proteins with their cellular receptors, including integrins. AII and other growth factors augment beta(1)-integrin-dependent adhesion and spreading by "inside-out signaling" without affecting the total number of integrin receptors. "Inside-out signaling" involves specific signaling pathways, including protein kinase C (PKC), leading to activation of beta1-integrins. In the present study we investigated the mechanisms involved in AII-increased adhesion of adult rat cardiac fibroblasts (CFBs), obtained from Sprague-Dawley rats, to collagen I mediated by beta1-integrin.

    Treatment of CFBs with AII induced a concentration-dependent increase in adhesion to collagen I (2.2-fold, p<0.01) within 3-6 h of treatment. This effect was mediated by beta1-integrin via the angiotensin AT1 receptor and was significantly reduced by protein kinase C inhibition. AII significantly induced phosphorylation of PKC epsilon (PKCepsilon), which is involved in beta1-integrin-dependent adhesion and motility, and phosphorylation of the cytoplasmatic tail of beta1-integrin at threonine 788/789, required for adhesion. Phosphorylation of beta1-integrin and an increase in adhesion was also induced by l-alpha-phosphatidylinositol-3,4,5-triphosphate (l-alpha-PIP3), an activator of endogenous PKCepsilon. AII failed to increase adhesion in myofibroblasts obtained from PKCepsilon (-/-) mice, but not in cells obtained from control mice. Co-immunoprecipitation and double immunofluorescence demonstrated that AII induced a close association of PKCepsilon with beta1-integrin in CFBs.

    Conclusion: The present study demonstrates that AII increased beta1-integrin-dependent adhesion to collagen I in cardiac fibroblasts by inside-out signaling via PKCepsilon and phosphorylation of the cytoplasmatic tail of the beta1-integrin.

    Cardiovascular research 2005;67;1;50-9

  • Cross-desensitization among CXCR1, CXCR2, and CCR5: role of protein kinase C-epsilon.

    Nasser MW, Marjoram RJ, Brown SL and Richardson RM

    Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, 27707, USA.

    The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKCalpha, -beta1, and -beta2 to the membrane, but only CXCR1 activated PKCepsilon. CXCL8 also failed to activate PKCepsilon in RBL-2H3 cells stably expressing CXCR2. DeltaCXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKCepsilon as well as CXCR1. Expression of the PKCepsilon inhibitor peptide epsilonV1 in RBL-2H3 cells blocked PKCepsilon translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization. epsilonV1 also inhibited CXCR1-, CCR5-, and DeltaCXCR2-mediated cross-regulatory signals for GTPase activity, Ca(2+) mobilization, and internalization. Peritoneal macrophages from PKCepsilon-deficient mice (PKCepsilon(-/-)) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca(2+) mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKCepsilon to mediate cross-inhibitory signals. Inhibition or deletion of PKCepsilon decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization, suggesting that cross-regulation is a Ca(2+)-independent process. Because DeltaCXCR2, but not CXCR2, activates PKCepsilon and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.

    Funded by: NCI NIH HHS: CA92077; NIAID NIH HHS: AI38910

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;11;6927-33

  • Cardioprotection mediated by urocortin is dependent on PKCepsilon activation.

    Lawrence KM, Kabir AM, Bellahcene M, Davidson S, Cao XB, McCormick J, Mesquita RA, Carroll CJ, Chanalaris A, Townsend PA, Hubank M, Stephanou A, Knight RA, Marber MS and Latchman DS

    Medical Molecular Biology Unit, Institute of Child Health, University College, London, UK. k.lawrence@ich.ucl.ac.uk

    Urocortin (Ucn) is an endogenous cardioprotective agent that protects against the damaging effects of ischemia and reperfusion injury in vitro and in vivo. We have found that the mechanism of action of Ucn involves both acute activation of specific target molecules, and using Affymetrix (Santa Clara, CA) gene chip technology, altered gene expression of different end effector molecules. Here, from our gene chip data, we show that after a 24 h exposure to Ucn, there was a specific increase in mRNA and protein levels of the protein kinase C epsilon (PKCepsilon) isozyme in primary rat cardiomyocytes compared with untreated cells and in the Langendorff perfused ex vivo heart. Furthermore, a short 10 min exposure of these cells to Ucn caused a specific translocation/activation of PKCepsilon in vitro and in the Langendorff perfused ex vivo heart. The importance of the PKCepsilon isozyme in cardioprotection and its relationship to cardioprotection produced by Ucn was assessed using PKCepsilon-specific inhibitor peptides. The inhibitor peptide, when introduced into cardiomyocytes, caused an increase in apoptotic cell death compared with control peptide after ischemia and reperfusion. When the inhibitor peptide was present with Ucn, the cardioprotective effect of Ucn was lost. This loss of cardioprotection by Ucn was also seen in whole hearts from PKCepsilon knockout mice. These findings indicate that the cardioprotective effect of Ucn is dependent upon PKCepsilon.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;7;831-3

  • Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation.

    Wang W, Jobbagy Z, Bird TH, Eiden MV and Anderson WB

    Laboratory of Cellular Oncology, NCI, National Institutes of Health, Bethesda, MD 20892, USA.

    Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

    The Journal of biological chemistry 2005;280;17;16772-83

  • Increased collagen deposition and diastolic dysfunction but preserved myocardial hypertrophy after pressure overload in mice lacking PKCepsilon.

    Klein G, Schaefer A, Hilfiker-Kleiner D, Oppermann D, Shukla P, Quint A, Podewski E, Hilfiker A, Schröder F, Leitges M and Drexler H

    Department of Cardiovascular Medicine, Hannover Medical School, Germany. gunnarklein@yahoo.de

    Overexpression and activation of protein kinase C-epsilon (PKCepsilon) results in myocardial hypertrophy. However, these observations do not establish that PKCepsilon is required for the development of myocardial hypertrophy. Thus, we subjected PKCepsilon-knockout (KO) mice to a hypertrophic stimulus by transverse aortic constriction (TAC). KO mice show normal cardiac morphology and function. TAC caused similar cardiac hypertrophy in KO and wild-type (WT) mice. However, KO mice developed more interstitial fibrosis and showed enhanced expression of collagen Ialpha1 and collagen III after TAC associated with diastolic dysfunction, as assessed by tissue Doppler echocardiography (Ea/Aa after TAC: WT 2.1+/-0.3 versus KO 1.0+/-0.2; P<0.05). To explore underlying mechanisms, we analyzed the left ventricular (LV) expression pattern of additional PKC isoforms (ie, PKCalpha, PKCbeta, and PKCdelta). After TAC, expression and activation of PKCdelta protein was increased in KO LVs. Moreover, KO LVs displayed enhanced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), whereas p42/p44-MAPK activation was attenuated. Under stretch, cultured KO fibroblasts showed a 2-fold increased collagen Ialpha1 (col Ialpha1) expression, which was prevented by PKCdelta inhibitor rottlerin or by p38 MAPK inhibitor SB 203580. In conclusion, PKCepsilon is not required for the development of a pressure overload-induced myocardial hypertrophy. Lack of PKCepsilon results in upregulation of PKCdelta and promotes activation of p38 MAPK and JNK, which appears to compensate for cardiac hypertrophy, but in turn, is associated with increased collagen deposition and impaired diastolic function.

    Circulation research 2005;96;7;748-55

  • Regulation of highly cytokinergic IgE-induced mast cell adhesion by Src, Syk, Tec, and protein kinase C family kinases.

    Kitaura J, Eto K, Kinoshita T, Kawakami Y, Leitges M, Lowell CA and Kawakami T

    Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

    Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Recent studies have shown that, contrary to the traditional view, binding of monomeric IgE to Fc epsilon RI results in a number of biological outcomes in mast cells, including survival. However, IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic (HC) IgEs cause extensive Fc epsilon RI aggregation, which leads to potent enhancement of survival and other activation events, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. The present study demonstrates that HC, but not PC, IgEs can efficiently induce adhesion and spreading of mouse mast cells on fibronectin-coated plates in slow and sustained kinetics. HC IgE-induced adhesion through beta1 and beta7 integrins promotes survival, IL-6 production, and DNA synthesis. Importantly, we have identified Lyn and Syk as requisite tyrosine kinases and Hck, Btk, and protein kinase C theta as contributory kinases in HC IgE-induced adhesion and spreading, whereas protein kinase C epsilon plays a negative role. Consistent with these results, Lyn, Syk, and Btk are activated in HC IgE-stimulated cells in a slower but more sustained manner, compared with cells stimulated with IgE and Ag. Thus, binding of HC IgEs to Fc epsilon RI induces adhesion of mast cells to fibronectin by modulating cellular activation signals in a unique fashion.

    Funded by: NIAID NIH HHS: AI50209, R01 AI038348-09, R01 AI050209-04; NIGMS NIH HHS: GM38348

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;8;4495-504

  • PKCepsilon activation augments cardiac mitochondrial respiratory post-anoxic reserve--a putative mechanism in PKCepsilon cardioprotection.

    McCarthy J, McLeod CJ, Minners J, Essop MF, Ping P and Sack MN

    Hatter Institute for Cardiology Research, University of Cape Town Medical School, Cape Town, South Africa.

    Modest cardiac-overexpression of constitutively active PKCepsilon (aPKCepsilon) in transgenic mice evokes cardioprotection against ischemia. As aPKCepsilon interacts with mitochondrial respiratory-chain proteins we hypothesized that aPKCepsilon modulates respiration to induce cardioprotection. Using isolated cardiac mitochondria wild-type and aPKCepsilon mice display similar basal mitochondrial respiration, rate of ATP synthesis and adenosine nucleotide translocase (ANT) functional content. Conversely, the aPKCepsilon mitochondria exhibit modest hyperpolarization of their inner mitochondrial membrane potential (DeltaPsi(m)) compared to wild-type mitochondrial by flow cytometry. To assess whether this hyperpolarization engenders resilience to simulated ischemia, anoxia-reoxygenation experiments were performed. Mitochondria were exposed to 45 min anoxia followed by reoxygenation. At reoxygenation, aPKCepsilon mitochondria recovered ADP-dependent respiration to 44 +/- 3% of baseline compared to 28 +/- 2% in WT controls (P = 0.03) in parallel with enhanced ATP synthesis. This preservation in oxidative phosphorylation is coupled to greater ANT functional content [42% > concentration of atractyloside for inhibition in the aPKCepsilon mitochondria vs. WT control (P < 0.0001)], retention of mitochondrial cytochrome c and conservation of DeltaPsi(m). These data demonstrate that mitochondria from PKCepsilon activated mice are intrinsically resilient to anoxia-reoxygenation compared to WT controls. This resilience is in part due to enhanced recovery of oxidative phosphorylation coupled to maintained ANT activity. As maintenance of ATP is a prerequisite for cellular viability we conclude that PKCepsilon activation augmented mitochondrial respiratory capacity in response to anoxia-reoxygenation may contribute to the PKCepsilon cardioprotective program.

    Journal of molecular and cellular cardiology 2005;38;4;697-700

  • Functional proteomic analysis of a three-tier PKCepsilon-Akt-eNOS signaling module in cardiac protection.

    Zhang J, Baines CP, Zong C, Cardwell EM, Wang G, Vondriska TM and Ping P

    Division of Cardiology, Departments of Physiology and Medicine, University of California at Los Angeles, Los Angeles, California, USA.

    Cardiac protective signaling networks have been shown to involve PKCepsilon. However, the molecular mechanisms by which PKCepsilon interacts with other members of these networks to form task-specific modules remain unknown. Among 93 different PKCepsilon-associated proteins that have been identified, Akt and endothelial nitric oxide (NO) synthase (eNOS) are of importance because of their independent abilities to promote cell survival and prevent cell death. The simultaneous association of PKCepsilon, Akt, and eNOS has not been examined, and, in particular, the formation of a module containing these three proteins and the role of such a module in the regulation of NO production and cardiac protection are unknown. The present study was undertaken to determine whether these molecules form a signaling module and, thereby, play a collective role in cardiac signaling. Using recombinant proteins in vitro and PKCepsilon transgenic mouse hearts, we demonstrate the following: 1) PKCepsilon, Akt, and eNOS interact and form signaling modules in vitro and in the mouse heart. Activation of either PKCepsilon or Akt enhances the formation of PKCepsilon-Akt-eNOS signaling modules. 2) PKCepsilon directly phosphorylates and enhances activation of Akt in vitro, and PKCepsilon activation increases phosphorylation and activation of Akt in PKCepsilon transgenic mouse hearts. 3) PKCepsilon directly phosphorylates eNOS in vitro, and this phosphorylation enhances eNOS activity. Activation of PKCepsilon in vivo increased phosphorylation of eNOS at Ser(1177), indicating eNOS activation. This study characterizes, for the first time, the physical, as well as functional, coupling of PKCepsilon, Akt, and eNOS in the heart and implicates these PKCepsilon-Akt-eNOS signaling modules as critical signaling elements during PKCepsilon-induced cardiac protection.

    Funded by: NHLBI NIH HHS: HL-65431, HL-69301

    American journal of physiology. Heart and circulatory physiology 2005;288;2;H954-61

  • Protein kinase Cepsilon is dispensable for TCR/CD3-signaling.

    Gruber T, Thuille N, Hermann-Kleiter N, Leitges M and Baier G

    Department for Medical Biology and Human Genetics, Medical University of Innsbruck, Schoepfstrasse 41, A-6020 Innsbruck, Austria.

    PKCepsilon has been strongly linked to cell activation and proliferation in many cell types, including leukemic T-cell lines. In particularly, an essential role of PKCepsilon has been established in the IKK-beta/I-kappaB/NF-kappaB transactivation cascade. To study the physiological function of PKCepsilon in primary T-cells, we used our newly established PKCepsilon null mice. Unexpectedly, however, we did not reveal any defect in the development and function of CD3+ T-cells. Proliferative responses as well as IL-2 cytokine secretion of PKCepsilon-deficient T-cells induced by allogenic MHC, plate-bound anti-CD3 antibodies (with or without anti-CD28 costimulation), or mitogenic stimuli such as phorbol ester and Ca2+ ionophore were comparable with wild-type controls. Consistently, after CD3/CD28 engagement, deficiency of PKCepsilon did not impair NF-kappaB transactivation as well as CD25, CD44 and CD69 induction. Thus, PKCepsilon-deficient T-cells had similar physiological thresholds for activation in vitro. This finding suggests that PKCepsilon plays a redundant role in TCR-induced regulation of T-cell proliferation.

    Molecular immunology 2005;42;3;305-10

  • Specific modulation of Na+ channels in hippocampal neurons by protein kinase C epsilon.

    Chen Y, Cantrell AR, Messing RO, Scheuer T and Catterall WA

    Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.

    Acetylcholine binding to muscarinic acetylcholine receptors activates G-proteins, phospholipase C, and protein kinase C (PKC), which phosphorylates brain Na+ channels and reduces peak Na+ current in hippocampal neurons. Because multiple PKC isozymes with different regulatory properties are expressed in hippocampal neurons, we investigated which ones are responsible for mediating this effect. The diacylglycerol analog oleoylacetylglycerol (OAG) reduced the amplitude of Na+ current in dissociated mouse hippocampal neurons by 28.5 +/- 5.3% (p < 0.01). The reduction of peak Na+ current was similar with Ca2+-free internal solution and in 92 nm internal Ca2+, suggesting that calcium-dependent, conventional PKC isozymes were unlikely to mediate this response. Gö6976, which inhibits conventional PKC isozymes, reduced the effect of PKC activators only slightly, whereas rottlerin, which inhibits PKCdelta preferentially at 5 microm, had no effect. Ro-31-8425 (20 nm), which inhibits conventional PKC isozymes, did not reduce the response to OAG. However, higher concentrations of Ro-31-8425 (100 nm or 1 microm) that inhibit novel PKC isozymes effectively blocked OAG inhibition of Na+ current. Inclusion of a selective PKCepsilon-anchoring inhibitor peptide (PKCepsilon-I) in the recording pipette prevented the reduction of peak Na+ current by OAG, whereas an anchoring inhibitor peptide specific for PKCbeta and an inactive scrambled PKCepsilon-I peptide had no effect. In addition, OAG had no effect on Na+ current in hippocampal neurons from PKCepsilon null mice. Overall, our data from four experimental approaches indicate that anchored PKCepsilon is the isozyme responsible for PKC-mediated reduction of peak Na+ currents in mouse hippocampal neurons.

    Funded by: NIMH NIH HHS: K01 MH01669; NINDS NIH HHS: R01 NS15751

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;2;507-13

  • Overexpression of protein kinase C-{epsilon} in the mouse epidermis leads to a spontaneous myeloproliferative-like disease.

    Wheeler DL, Reddig PJ, Ness KJ, Leith CP, Oberley TD and Verma AK

    Department of Human Oncology, Medical School, University of Wisconsin, K4/532 CSC Clinical Science Center, 600 Highland Ave., Madison, WI 53792, USA.

    Protein kinase C (PKC)-epsilon, a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mouse lines that overexpress (8- or 18-fold) PKC-epsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate-promotion or by repeated ultraviolet radiation exposures. The susceptibility to the development of SCC was proportional to the level of expression of the PKC-epsilon transgene. We now report that PKC-epsilon FVB/N transgenic mice (line 215) that overexpress in epidermis approximately 18-fold PKC-epsilon protein more than their wild-type littermates spontaneously develop a myeloproliferative-like disease (MPD) in 100% of PKC-epsilon transgenic mice. The MPD was characterized by an excess of neutrophils and eosinophils, resulting in invasion of almost all vital organs of the mouse by 6 months of age. On gross examination these mice present with splenomegaly, hepatomegaly, and severe lymphadenopathy. Examination of the bone marrow revealed almost complete effacement by neutrophils, eosinophils, and their precursors. Furthermore, the spleen and lymph nodes were enlarged and exhibited marked extramedullary hematopoiesis. Complete pathological analysis of the second PKC-epsilon transgenic mouse (line 224) that expresses approximately eightfold PKC-epsilon protein more than their wild-type littermates revealed no remarkable findings in any of the affected organs as seen in line 215. However, peripheral blood analyses of PKC-epsilon transgenic mice indicated significant increases of neutrophils in the circulating blood in both PKC-epsilon transgenic lines. To determine whether there was an imbalance of cytokines in PKC-epsilon transgenic mice (line 215), resulting in aberrant myelopoiesis, we analyzed 17 cytokines in the peripheral blood. This analysis indicated that interleukin-5, interleukin-6, and granulocyte-colony stimulating factor were up-regulated as a function of age. The transgene PKC-epsilon was not detected in any of the affected organs (bone marrow, liver, spleen, lung) We suggest that overexpression of PKC-epsilon in the epidermis may lead to the induction of specific cytokines that may, in a paracrine mechanism, perturb normal hematopoiesis in bone marrow resulting in a granulocytic skew toward that of neutrophils and eosinophils. The susceptibility of PKC-epsilon transgenic mice to the induction of SCC and the spontaneous development of MPD are unrelated.

    Funded by: NCI NIH HHS: CA 35368

    The American journal of pathology 2005;166;1;117-26

  • Protein kinase C activity regulates the onset of anaphase I in mouse oocytes.

    Viveiros MM, O'brien M and Eppig JJ

    Center for Animal Transgenesis and Germ Cell Research, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348, USA. viveiros@vet.upenn.edu

    The metaphase-to-anaphase I transition is a key step in the completion of meiosis I. In mouse oocytes, competence to exit metaphase I (MI) is developmentally regulated and typically not acquired until the preovulatory stage. The possible role of protein kinase C (PKC) in regulating this critical transition was assessed in both normal oocytes isolated from small antral follicles (18-day-old B6SJLF1 mice), which have not yet developed the capacity to progress to metaphase II (MII), and also oocytes defective in their ability to exit MI despite development to the preovulatory stage (24-day-old CX8 recombinant inbred strains). In both systems, transient suppression of endogenous PKC activity by treatment with a PKC-specific inhibitor, bisindolylmaleimide I (BIM), promoted the onset of anaphase I in a dose-dependent manner, while activation of PKC with the phorbol ester TPA blocked progression to MII. Following a 2-h incubation with BIM, the majority of oocytes progressed to, and arrested at, MII. The resulting MII oocytes were fertilizable in vitro, showing similar cleavage and blastocyst development rates between BIM treated and untreated controls. Transferred embryos resulted in the development of pups to term in both groups. These data demonstrate that PKC plays an important role in regulating the onset of anaphase I in mouse oocytes. Moreover, it is concluded that oocytes isolated from small antral follicles become blocked at MI due to a PKC-mediated signal, suggesting that acquisition of competence to complete meiosis I involves, in part, the control of PKC activity. Similarly, failure to regulate PKC activity at the preovulatory stage likely promotes arrest at MI.

    Funded by: NCI NIH HHS: CA 34196, CA 62392; NICHD NIH HHS: HD21970

    Biology of reproduction 2004;71;5;1525-32

  • Sphingosine kinase activation mediates ischemic preconditioning in murine heart.

    Jin ZQ, Goetzl EJ and Karliner JS

    Cardiology Section, VA Medical Center and Department of Medicine, University of California, San Francisco, USA.

    Background: Phosphorylation of sphingosine by sphingosine kinase (SK) is the rate-limiting step in the cellular synthesis of sphingosine 1-phosphate (S1P). The monoganglioside GM1, which stimulates SK, is cardioprotective in part through increased generation of S1P that protects myocytes by diverse mechanisms. Because protein kinase C (PKC)epsilon activation is necessary for myocardial ischemic preconditioning (IPC) and PKC activators increase SK activity, we tested the hypothesis that SK may be a central mediator of IPC.

    In adult murine hearts, IPC sufficient to reduce infarct size significantly increased cardiac SK activity, induced translocation of SK protein from the cytosol to membranes, and enhanced cardiac myocyte survival. IPC did not increase SK activity in PKCepsilon-null mice. The SK antagonist N,N-dimethylsphingosine inhibited PKCepsilon activation and directly abolished the protective effects of IPC and the enhanced SK activity induced by IPC.

    Conclusions: These findings demonstrate that PKCepsilon is thus recruited by IPC and induces activation of SK that then mediates IPC-induced cardioprotection in murine heart.

    Funded by: NHLBI NIH HHS: 1P01HL068738-01A1

    Circulation 2004;110;14;1980-9

  • Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia.

    Ueyama T, Lennartz MR, Noda Y, Kobayashi T, Shirai Y, Rikitake K, Yamasaki T, Hayashi S, Sakai N, Seguchi H, Sawada M, Sumimoto H and Saito N

    Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan.

    Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by Gö6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.

    Journal of immunology (Baltimore, Md. : 1950) 2004;173;7;4582-9

  • Activation of protein kinase C epsilon inhibits the two-pore domain K+ channel, TASK-1, inducing repolarization abnormalities in cardiac ventricular myocytes.

    Besana A, Barbuti A, Tateyama MA, Symes AJ, Robinson RB and Feinmark SJ

    Center for Molecular Therapeutics, Department of Pharmacology, Columbia University, New York, New York 10032, USA.

    Activation of the platelet-activating factor (PAF) receptor leads to a decrease in outward current in murine ventricular myocytes by inhibiting the TASK-1 channel. TASK-1 carries a background or "leak" current and is a member of the two-pore domain potassium channel family. Its inhibition is sufficient to delay repolarization, causing prolongation of the action potential duration, and in some cases, early after depolarizations. We set out to determine the cellular mechanisms that control regulation of TASK-1 by PAF. Inhibition of TASK-1 via activation of the PAF receptor is protein kinase C (PKC)-dependent. Using isoform-specific PKC inhibitor or activator peptides in patch clamp experiments, we now demonstrate that activation of PKCepsilon is both necessary and sufficient to regulate murine TASK-1 current in a heterologous expression system and to induce repolarization abnormalities in isolated myocytes. Furthermore, site-directed mutagenesis studies have identified threonine 381, in the C-terminal tail of murine TASK-1, as a critical residue in this regulation.

    Funded by: NHLBI NIH HHS: HL70105

    The Journal of biological chemistry 2004;279;32;33154-60

  • Role for PKC-epsilon in neuronal death induced by oxidative stress.

    Jung YS, Ryu BR, Lee BK, Mook-Jung I, Kim SU, Lee SH, Baik EJ and Moon CH

    Department of Physiology, School of Medicine, Ajou University, Suwon, Kyungkido 442-749, Republic of Korea.

    We investigated which isoforms of PKCs can be modulated and what their roles are during l-buthionine-S,R-sulfoximine (BSO)-induced neuronal death. We observed the isoform specific translocation of PKC-epsilon from the soluble fraction to the particulate in cortical neurons treated with 10 mM BSO. The translocation of PKC-epsilon by BSO was blocked by antioxidant trolox, suggesting the PKC-epsilon as a downstream of reactive oxygen species (ROS) elevated by BSO. Trolox inhibited the ROS elevation and the neuronal death in BSO-treated cortical cells. The BSO-induced neuronal death was remarkably inhibited by both the pharmacological inhibition of PKC-epsilon with epsilonV1-2 and the functional blockade for PKC-epsilon through overexpression of PKC-epsilon V1 region, suggesting the detrimental role of PKC-epsilon. These results suggest that PKC-epsilon is the major PKC isoform involved in the pathways triggered by ROS, leading to neuronal death in BSO-treated cortical neurons.

    Biochemical and biophysical research communications 2004;320;3;789-94

  • Phorbol ester-induced translocation of PKC epsilon to the nucleus in fibroblasts: identification of nuclear PKC epsilon-associating proteins.

    Xu TR and Rumsby MG

    Department of Biology, University of York, York YO10 5DD, UK.

    We show that phorbol ester treatment of NIH 3T3 fibroblasts induces rapid translocation of PKC from a perinuclear site to the nucleus, extending findings in PC12 and NG108-15 cells and in myocytes. We have immunoprecipitated the PKC from nuclei isolated from phorbol ester-treated fibroblasts and identified six proteins which associate with nuclear PKC. These have been characterised as matrin 3, transferrin, Rac GTPase activating protein 1, vimentin, beta-actin and annexin II by MALDI-TOF-MS. We have confirmed that these proteins associate with PKC by gel overlay and/or dot blotting assays. The role of these PKC-associating proteins in the nucleus and their interaction with PKC are considered.

    FEBS letters 2004;570;1-3;20-4

  • Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo.

    Roman BB, Goldspink PH, Spaite E, Urboniene D, McKinney R, Geenen DL, Solaro RJ and Buttrick PM

    Section of Cardiology, University of Illinois, 840 S. Wood Street (M/C 715), Chicago, IL 60612, USA. broman@uic.edu

    Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-epsilon, may slow the rates of cardiac contraction and relaxation (+dP/dt and -dP/dt). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser(43) and Ser(45) and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater -dP/dt than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/dt increased by 33% and -dP/dt increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.

    Funded by: NHLBI NIH HHS: P01 HL 62426, R01 HL 0609961-01, R3L HL 22231

    American journal of physiology. Heart and circulatory physiology 2004;286;6;H2089-95

  • Sublethal simulated ischemia promotes delayed resistance against ischemia via ATP-sensitive (K+) channels in murine myocytes: role of PKC and iNOS.

    Nayeem MA

    DIR ETP LPC MEM, National Institute of Environment and Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. nayeem@niehs.nih.gov

    In this study, we examined whether sublethal simulated ischemia (SSI) induces delayed cellular protection in mouse cardiac myocytes, and whether the delayed cellular protection depends on the activation of protein kinase C-epsilon (PKC-epsilon), inducible nitric oxide synthase (iNOS), and ATP-sensitive K(+) (K(ATP)) channels against subsequent sustained simulated ischemia (SI). The following groups of mouse cardiac myocytes were studied: (a) SI: incubation with SI buffer for 1 h; (b) SSI: incubation with SSI buffer for 30 min; (c) SSI + PKC inhibitor, chelerythrine chloride (CCl): SSI and 1 micro M CCl; (d) SSI + iNOS inhibitor, S-methylthiourea (SMT): SSI and 100 nM SMT; (e) SSI + K(ATP) channel blocker, glibenclamide (Glb): SSI and 50 micro M Glb; (f) SSI + mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD): SSI and 50 micro M 5-HD. The release of lactate dehydrogenase into the medium and the amount remaining in the cells was measured, and A(1) adenosine receptor, PKC-epsilon, and iNOS were detected through western blot analysis. The delayed cellular protection acquired due to SSI showed a decreased release of lactate dehydrogenase (%) from 46.51 +/- 1.60 to 37.00 +/- 1.34 (p < 0.001) and was blocked by CCl (47.08 +/- 0.95), SMT (48.08 +/- 1.18), Glb (45.88 +/- 1.31), and 5-HD (47.20 +/- 1.56). Simultaneously, SSI-induced up-regulation of A(1) adenosine receptor, PKC-epsilon, iNOS, and opening of both membrane and mitochondrial K(ATP) channels also was observed compared with controls.

    Antioxidants & redox signaling 2004;6;2;375-83

  • Increased particulate partitioning of PKC epsilon reverses susceptibility of phospholamban knockout hearts to ischemic injury.

    Gregory KN, Hahn H, Haghighi K, Marreez Y, Odley A, Dorn GW and Kranias EG

    Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267 0575, USA.

    Cytosolic Ca(2+) overload is a critical mediator of myocardial damage following cardiac ischemia-reperfusion. It has therefore been proposed that normalization of sarcoplasmic reticulum Ca(2+) cycling through inhibition or ablation of the Ca(2+) ATP-ase inhibitor phospholamban (PLN), which shows promise as a treatment for heart failure, could be beneficial in ischemic heart disease. However, a recent study has shown that globally ischemic PLN-deficient hearts exhibit increased ischemic injury, with impaired contractile, ATP, and phosphocreatine recoveries, compared to wild-type hearts. Since protein kinase C (PKC) family members are widely recognized as mediators of both post-ischemic injury and ischemic preconditioning, we assessed PKC levels in PLN-deficient hearts. Compared to genetically normal hearts, PLN-deficient hearts exhibited diminished particulate partitioning of PKC, a known cardioprotective PKC isoform, without alterations in the levels of membrane-associated PKC delta nor PKC alpha. To determine if decreased particulate partitioning of cardioprotective PKC epsilon was a cause of increased ischemic injury in PLN-deficient hearts, PLN-deficient mice were mated with mice expressing a myocardial-specific PKC epsilon translocation activator peptide, pseudo-epsilon receptor for activated kinase C (psi epsilon RACK). In psi epsilon RACK/PLN knockout (KO) hearts, PKC epsilon translocation to membranous cellular structures was augmented and this was associated with a significant acceleration of post-ischemic contraction and relaxation rates, as well as reduction of creatine phosphokinase release, compared to PLN-deficient hearts. Importantly, post-ischemic functional recovery reached pre-ischemic hyperdynamic values in psi epsilon RACK/PLN KO hearts, indicating super-rescue by the combination of PLN ablation and psi epsilon RACK expression. These findings suggest that diminished PKC epsilon particulate partitioning in PLN-deficient hearts is associated with attenuated contractile recovery upon ischemia-reperfusion and that increased translocation of PKC to membranous cellular structures confers full cardioprotection.

    Funded by: NHLBI NIH HHS: HL-26057, HL-52318, HL-58010, HL-59888, HL-64018

    Journal of molecular and cellular cardiology 2004;36;2;313-8

  • Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders.

    Aksoy E, Goldman M and Willems F

    Laboratory of Experimental Immunology, Université Libre de Bruxelles, B-1070, Brussels, Belgium

    Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by a set of germline-encoded receptors: the Toll-like receptors (TLRs). TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity. Indeed, activation of T lymphocytes depends on their interaction with dendritic cells previously stimulated by TLR agonists such as bacterial lipopolysaccharide (LPS), a TLR-4 ligand. A novel PKC epsilon (epsilon) was recently found to be a critical component of TLR-4 signaling pathway and thereby to play a key role in macrophage and dendritic cell (DC) activation in response to LPS. Thus, controlling the kinase activity of PKC epsilon might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin.

    The international journal of biochemistry & cell biology 2004;36;2;183-8

  • Preservation of base-line hemodynamic function and loss of inducible cardioprotection in adult mice lacking protein kinase C epsilon.

    Gray MO, Zhou HZ, Schafhalter-Zoppoth I, Zhu P, Mochly-Rosen D and Messing RO

    Department of Medicine, University of California, San Francisco, California 94110, USA. gray@medicine.ucsf.edu

    Signaling pathways involving protein kinase C isozymes are modulators of cardiovascular development and response to injury. Protein kinase C epsilon activation in cardiac myocytes reduces necrosis caused by coronary artery disease. However, it is unclear whether protein kinase C epsilon function is required for normal cardiac development or inducible protection against oxidative stress. Protein kinase C delta activation is also observed during cardiac preconditioning. However, its role as a promoter or inhibitor of injury is controversial. We examined hearts from protein kinase C epsilon knock-out mice under physiological conditions and during acute ischemia reperfusion. Null-mutant and wild-type mice displayed equivalent base-line morphology and hemodynamic function. Targeted disruption of the protein kinase C epsilon gene blocked cardioprotection caused by ischemic preconditioning and alpha(1)-adrenergic receptor stimulation. Protein kinase C delta activation increased in protein kinase C epsilon knock-out myocytes without altering resistance to injury. These observations support protein kinase C epsilon activation as an essential component of cardioprotective signaling. Our results favor protein kinase C delta activation as a mediator of normal growth. This study advances the understanding of cellular mechanisms responsible for preservation of myocardial integrity as potential targets for prevention and treatment of ischemic heart disease.

    Funded by: NIAAA NIH HHS: 5R01AA011135; NIGMS NIH HHS: 5T32GM007546

    The Journal of biological chemistry 2004;279;5;3596-604

  • Specific isoforms of protein kinase C are essential for prevention of folate-resistant neural tube defects by inositol.

    Cogram P, Hynes A, Dunlevy LP, Greene ND and Copp AJ

    Neural Development Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

    A proportion of neural tube defects (NTDs) can be prevented by maternal folic acid supplementation, although some cases are unresponsive. The curly tail mutant mouse provides a model of folate-resistant NTDs, in which defects can be prevented by inositol therapy in early pregnancy. Hence, inositol represents a possible novel adjunct therapy to prevent human NTDs. The present study investigated the molecular mechanism by which inositol prevents mouse NTDs. Activation of protein kinase C (PKC) is known to be essential, and we examined neurulation-stage embryos for PKC expression and applied PKC inhibitors to curly tail embryos developing in culture. Although all known PKC isoforms were detected in the closing neural tube, use of chemical PKC inhibitors identified a particular requirement for 'conventional' PKC isoforms. Peptide inhibitors offer selective inhibition of individual PKCs, and we demonstrated isoform-specific inhibition of PKC in embryonic cell cultures. Application of peptide inhibitors to neurulation-stage embryos revealed an absolute dependence on the activity of PKCbetaI and gamma for prevention of NTDs by inositol, and partial dependence on PKCzeta, whereas other PKCs (alpha, betaII delta, and epsilon) were dispensable. To investigate the cellular action of inositol and PKCs in NTD prevention, we examined cell proliferation in curly tail embryos. Defective proliferation of hindgut cells is a key component of the pathogenic sequence leading to NTDs in curly tail. Hindgut cell proliferation was stimulated specifically by inositol, an effect that required activation of PKCbetaI. Our findings reveal an essential role of specific PKC isoforms in mediating the prevention of mouse NTDs by inositol.

    Human molecular genetics 2004;13;1;7-14

  • Differential implication of protein kinase C isoforms in cytotoxic T lymphocyte degranulation and TCR-induced Fas ligand expression.

    Pardo J, Buferne M, Martínez-Lorenzo MJ, Naval J, Schmitt-Verhulst AM, Boyer C and Anel A

    Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.

    CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.

    International immunology 2003;15;12;1441-50

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • Rapid association of protein kinase C-epsilon with insulin granules is essential for insulin exocytosis.

    Mendez CF, Leibiger IB, Leibiger B, Høy M, Gromada J, Berggren PO and Bertorello AM

    Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, SE-171 76 Stockholm, Sweden.

    Glucose-dependent exocytosis of insulin requires activation of protein kinase C (PKC). However, because of the great variety of isoforms and their ubiquitous distribution within the beta-cell, it is difficult to predict the importance of a particular isoform and its mode of action. Previous data revealed that two PKC isoforms (alpha and epsilon) translocate to membranes in response to glucose (Zaitzev, S. V., Efendic, S., Arkhammar, P., Bertorello, A. M., and Berggren, P. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9712-9716). Using confocal microscopy, we have now established that in response to glucose, PKC-epsilon but not PKC-alpha associates with insulin granules and that green fluorescent protein-tagged PKC-epsilon changes its distribution within the cell periphery upon stimulation of beta-cells with glucose. Definite evidence of PKC-epsilon requirement during insulin granule exocytosis was obtained by using a dominant negative mutant of this isoform. The presence of this mutant abolished glucose-induced insulin secretion, whereas transient expression of the wild-type PKC-epsilon led to a significant increase in insulin exocytosis. These results suggest that association of PKC-epsilon with insulin granule membranes represents an important component of the secretory network because it is essential for insulin exocytosis in response to glucose.

    Funded by: NIDDK NIH HHS: DK-58508

    The Journal of biological chemistry 2003;278;45;44753-7

  • Protein kinase Cepsilon is linked to 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor-alpha ectodomain shedding and the development of metastatic squamous cell carcinoma in protein kinase Cepsilon transgenic mice.

    Wheeler DL, Ness KJ, Oberley TD and Verma AK

    Department of Human Oncology, Medical School, University of Wisconsin, Madison, WI 53792, USA.

    Protein kinase Cepsilon (PKCepsilon), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately 18-fold) PKCepsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent metastatic squamous cell carcinoma (mSCC) elicited by 7,12-dimethylbenz(a)anthracene-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. We now present that PKCepsilon transgenic mice elicit elevated serum tumor necrosis factor (TNF)alpha levels during skin tumor promotion by TPA, and this increase may be linked to the development of mSCC. A single topical application of TPA (5 nmol) to the skin, as early as 2.5 h after treatment, resulted in a significant (P < 0.01) increase (2-fold) in epidermal TNFalpha and more than a 6-fold increase in ectodomain shedding of TNFalpha into the serum of PKCepsilon transgenic mice relative to their wild-type littermates. Furthermore, this TPA-stimulated TNFalpha shedding was proportional to the level of expression of PKCepsilon in the epidermis. Using the TNF-alpha converting enzyme (TACE) inhibitor, TAPI-1, TPA-stimulated TNFalpha shedding could be completely prevented in PKCepsilon transgenic mice and isolated keratinocytes. These results indicate that PKCepsilon signal transduction pathways to TPA-stimulated TNFalpha ectodomain shedding are mediated by TACE, a transmembrane metalloprotease. Using the superoxide dismutase mimetic CuDIPs and the glutathione reductase mimetic ebselen, TPA-stimulated TNFalpha shedding from PKCepsilon transgenic mice could be completely attenuated, implying the role of reactive oxygen species. Finally, i.p. injection of a TNFalpha synthesis inhibitor, pentoxifylline, during skin tumor promotion completely prevented the development of mSCC in PKCepsilon transgenic mice. Taken together, these results indicate that: (a) PKCepsilon activation is an initial signal in TPA-induced shedding of TNFalpha from epidermal keratinocytes; (b) PKCepsilon-mediated signals to TACE are possibly mediated through reactive oxygen species; and (c) TPA-induced TNFalpha shedding may play a role in the development of mSCC in PKCepsilon transgenic mice.

    Funded by: NCI NIH HHS: CA 35368

    Cancer research 2003;63;19;6547-55

  • Involvement of protein kinase C-epsilon in inositol hexakisphosphate-induced exocytosis in mouse pancreatic beta-cells.

    Høy M, Berggren PO and Gromada J

    Laboratory of Islet Cell Physiology, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark.

    Inositolhexakisphosphate (InsP6) plays a pivotal role in the pancreatic beta-cell stimulus-secretion coupling. We have used capacitance measurements to study the effects of InsP6 on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. In the presence of inhibitors of the protein phosphatase calcineurin to block endocytosis, intracellular application of InsP6 produced a dose-dependent stimulation of exocytosis, and half-maximal effect was observed at 22 microM. The stimulatory effect of InsP6 was dependent on protein kinase C (PKC) activity. Antisense oligonucleotides directed against specific PKC isoforms (alpha, beta II, delta, epsilon, xi) revealed the involvement of PKC-epsilon in InsP6-induced exocytosis. Furthermore, expression of dominant negative PKC-epsilon abolished InsP6-evoked exocytosis, whereas expression of wild-type PKC-epsilon led to a significant stimulation of InsP6-induced exocytosis. These data demonstrate that PKC-epsilon is involved in InsP6-induced exocytosis in pancreatic beta-cells.

    Funded by: NIDDK NIH HHS: DK 58508

    The Journal of biological chemistry 2003;278;37;35168-71

  • Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes.

    Soh JW and Weinstein IB

    Department of Medicine and Herbert Irving Comprehensive Cancer Center, College of Physicians & Surgeons, Columbia University, New York, New York 10032, USA.

    Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (Delta NPS and CAT), and dominant negative mutants of PKCs alpha, beta 1, beta 2, gamma, delta, epsilon, eta, zeta, and iota. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-alpha and PKC-epsilon were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-delta inhibited its activity. Transient transfection studies with a series of 5'-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-alpha. Deletion of the AP-1 enhancer element located at position -954 upstream from the initiation site abolished PKC-alpha-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-alpha and PKC-epsilon. The constitutively active mutants of PKC-alpha and PKC-epsilon also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-alpha or PKC-epsilon displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-alpha or PKC-epsilon in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation.

    Funded by: NCI NIH HHS: CA-26056

    The Journal of biological chemistry 2003;278;36;34709-16

  • Protein kinase C epsilon dependence of the recovery from down-regulation of S1P1 G protein-coupled receptors of T lymphocytes.

    Graeler MH, Kong Y, Karliner JS and Goetzl EJ

    Department of Medicine, University of California, San Francisco, California 94143-0711, USA.

    Sphingosine 1-phosphate (S1P) from mononuclear phagocytes and platelets signals T cells predominantly through S1P1 G protein-coupled receptors (Rs) to enhance survival, stimulate and suppress migration, and inhibit other immunologically relevant responses. Cellular S1P1 Rs and their signaling functions are rapidly down-regulated by S1P, through a protein kinase C (PKC)-independent mechanism, but characteristics of cell-surface re-expression of down-regulated S1P1 Rs have not been elucidated. T cell chemotactic responses (CT) to 10 and 100 nm S1P and inhibition of T cell chemotaxis to chemokines (CI) by 1 and 3 microm S1P were suppressed after 1 h of preincubation with 100 nm S1P, but recovered fully after 12-24 h of exposure to S1P. Late recovery of down-regulated CT and CI, but not early down-regulation, was suppressed by PKC and PKCepsilon-selective inhibitors and was absent in T cells from PKCepsilon-null mice. The same PKCepsilon inhibitors blocked S1P-evoked increases in T cell nuclear levels of c-Fos and phosphorylated c-Jun and JunD after 24 h, but not 1 h. A mixture of c-Fos plus c-Jun antisense oligonucleotides prevented late recovery of down-regulated CT and CI, without affecting S1P induction of down-regulation. Similarly, S1P-elicited threonine phosphorylation of S1P1 Rs was suppressed by a selective inhibitor of PKCepsilon after 24 h, but not 1 h. Biochemical requisites for recovery of down-regulated S1P1 Rs thus differ from those for S1P induction of down-regulation.

    Funded by: NHLBI NIH HHS: HL-31809, HL-68738

    The Journal of biological chemistry 2003;278;30;27737-41

  • Inhibition of the development of metastatic squamous cell carcinoma in protein kinase C epsilon transgenic mice by alpha-difluoromethylornithine accompanied by marked hair follicle degeneration and hair loss.

    Wheeler DL, Ness KJ, Oberley TD and Verma AK

    Department of Human Oncology, Medical School, University of Wisconsin, Madison, Wisconsin 53762, USA.

    The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C epsilon (PKC epsilon) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3-4-fold more in PKC epsilon transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC epsilon transgenic mice was completely prevented by administration of the suicide inhibitor of ODC alpha-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC epsilon transgenic mice. DFMO treatment-associated hair loss in PKC epsilon transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC epsilon transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC epsilon transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.

    Cancer research 2003;63;12;3037-42

  • EGF-stimulated signaling by means of PI3K, PLCgamma1, and PKC isozymes regulates branching morphogenesis of the fetal mouse submandibular gland.

    Koyama N, Kashimata M, Sakashita H, Sakagami H and Gresik EW

    Department of Cell Biology and Anatomical Sciences, The Sophie Davis School of Biomedical Education, The City University of New York Medical School, New York, New York 10031, USA.

    Branching morphogenesis of fetal mouse submandibular glands (SMGs) partly depends on the epidermal growth factor (EGF) receptor that triggers at least three intracellular signaling pathways involving (1) the mitogen-activated protein kinases ERK-1/2, (2) phospholipase Cgamma1 (PLCgamma1), and (3) phosphatidylinositol-3-kinase (PI3K). PLCgamma1 directly activates protein kinase C (PKC) isozymes; PI3K stimulates protein kinase B (PKB, also known as Akt), which ultimately activates PKCs and other proteins. We reported that the pattern of phosphorylation of ERK-1/2 in response to EGF in SMGs varies with fetal age and that blockade of EGF-stimulated ERK-1/2 signaling partially inhibits branching (Kashimata et al. [2000] Dev. Biol. 220:183-196). Here, we report on components of the PLCgamma1, PI3K, and PKC families of signaling molecules in fetal SMGs from the 13th day of gestation to postnatal ages. Western blotting revealed that (1) PLCgamma1 is present from E13 to E18 but drops off precipitously to negligible levels on the day of birth and thereafter, and (2) PI3K, PKB(Akt), and several PKC isozymes are expressed from E13 onward through adult life. Both PLCgamma1 and PI3K are phosphorylated in response to EGF. Inhibition of PI3K by LY294002 inhibited EGF-stimulated branching, but inhibition of PLCgamma1 by U73122 had no effect. Western blotting showed that the concentrations of 8 PKC isozymes vary with age in the fetal and postnatal SMG. However, general inhibition of PKCs by Calphostin C or specific inhibition of PKCalpha or of PKCepsilon by Gö6976 or Ro-32-0432, respectively, increased EGF-stimulated branching. Calphostin C also increased EGF-stimulated phosphorylation of ERK-1/2. These findings indicate that signaling from the EGF receptor in the fetal mouse SMG varies with development and triggers stimulatory effects by means of ERK-1/2 and PI3K but inhibitory effects by means of PKC isozymes.

    Funded by: NIDCR NIH HHS: DE10858

    Developmental dynamics : an official publication of the American Association of Anatomists 2003;227;2;216-26

  • Ethanol differentially enhances hippocampal GABA A receptor-mediated responses in protein kinase C gamma (PKC gamma) and PKC epsilon null mice.

    Proctor WR, Poelchen W, Bowers BJ, Wehner JM, Messing RO and Dunwiddie TV

    Department of Veterans Affairs Medical Center, Research Service, Denver, Colorado, USA. bill.proctor@uchsc.edu

    Ethanol intoxication results partly from actions of ethanol at specific ligand-gated ion channels. One such channel is the GABA(A) receptor complex, although ethanol's effects on GABA(A) receptors are variable. For example, we found that hippocampal neurons from selectively bred mice and rats with high hypnotic sensitivity to ethanol have increased GABA(A) receptor-mediated synaptic responses during acute ethanol treatment compared with mice and rats that display low behavioral sensitivity to ethanol. Here we investigate whether specific protein kinase C (PKC) isozymes modulate hypnotic and GABA(A) receptor sensitivity to ethanol. We examined acute effects of ethanol on GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) in mice lacking either PKCgamma (PKCgamma(-/-)) or PKCepsilon (PKCepsilon(-/-)) isozymes and compared the results to those from corresponding wild-type littermates (PKCgamma(+/+) and PKCepsilon(+/+)). GABA(A) receptor-mediated IPSCs were evoked in CA1 pyramidal neurons by electrical stimulation in stratum pyramidale, and the responses were recorded in voltage-clamp mode using whole-cell patch recording techniques. Ethanol (80 mM) enhanced the IPSC response amplitude and area in PKCgamma(+/+) mice, but not in the PKCgamma(-/-) mice. In contrast, ethanol markedly potentiated IPSCs in the PKCepsilon(-/-) mice, but not in PKCepsilon(+/+) littermates. There was a positive correlation between ethanol potentiation of IPSCs and the ethanol-induced loss of righting reflex such that mice with larger ethanol-induced increases in GABA(A) receptor-mediated IPSCs also had higher hypnotic sensitivity to ethanol. These results suggest that PKCgamma and PKCepsilon signaling pathways reciprocally modulate both ethanol enhancement of GABA(A) receptor function and hypnotic sensitivity to ethanol.

    Funded by: NIAAA NIH HHS: AA00141, AA03527, AA11275, AA13588

    The Journal of pharmacology and experimental therapeutics 2003;305;1;264-70

  • PKCepsilon is a permissive link in integrin-dependent IFN-gamma signalling that facilitates JAK phosphorylation of STAT1.

    Ivaska J, Bosca L and Parker PJ

    Protein Phosphorylation Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

    The critical dependence of receptor-triggered signals on integrin-mediated cell-substrate interactions represents a fundamental biological paradigm in health and disease. However, the molecular connections of these permissive inputs, which operate through integrin-matrix interactions, has remained largely obscure. Here we show that the serine-threonine kinase protein kinase C epsilon (PKCepsilon) functions as a signal integrator between cytokine and integrin signalling pathways. Integrins are shown to control PKCepsilon phosphorylation acutely by determining complex formation with protein phosphatase 2A (PP2A) and the upstream kinase PDK1 (phosphoinositide-dependent kinase 1). The PP2A-induced loss of PKCepsilon function results in attenuated interferon gamma (INF-gamma)-induced phosphorylation of STAT1 (signal transducer and activator of transcription 1) downstream of Janus kinase 1/2 (JAK1/2). PKCepsilon function and the IFN-gamma response can be recovered by inhibition of PP2A if PDK1 is associated with PKCepsilon in this complex. More directly, a PP2A-resistant mutant of PKCepsilon is sufficient for restoration of the IFN-gamma response in suspension culture. Thus, PKCepsilon functions as a central point of integration through which integrin engagement exerts a permissive input on IFN-gamma signalling.

    Nature cell biology 2003;5;4;363-9

  • Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction.

    Littler CM, Morris KG, Fagan KA, McMurtry IF, Messing RO and Dempsey EC

    Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

    PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-epsilon is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-epsilon would decrease acute HPV in adult C57BL6xSV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O(2)) was compared with responses to angiotensin II (ANG II) and KCl. PKC-epsilon -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-l-arginine augmented HPV in PKC-epsilon +/+ but not -/- mice. Inhibition of Ca(2+)-gated K(+) channels (K(Ca)) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K(+) channel (K(V)) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased K(V) channel expression could contribute to blunted HPV in PKC-epsilon -/- mice. Therefore, expression of the O(2)-sensitive K(V) channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Kv3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-epsilon blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or K(Ca) channel activity. Instead, the absence of PKC-epsilon allows increased expression of K(V) channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.

    Funded by: OAPP OPHS HHS: PPG-HL-14985

    American journal of physiology. Heart and circulatory physiology 2003;284;4;H1321-31

  • Phosphorylation or glutamic acid substitution at protein kinase C sites on cardiac troponin I differentially depress myofilament tension and shortening velocity.

    Burkart EM, Sumandea MP, Kobayashi T, Nili M, Martin AF, Homsher E and Solaro RJ

    University of Illinois at Chicago, Department of Physiology and Biophysics, Program in Cardiovascular Sciences, College of Medicine, Chicago, Illinois 60612, USA.

    There is evidence that multi-site phosphorylation of cardiac troponin I (cTnI) by protein kinase C is important in both long- and short-term regulation of cardiac function. To determine the specific functional effects of these phosphorylation sites (Ser-43, Ser-45, and Thr-144), we measured tension and sliding speed of thin filaments in reconstituted preparations in which endogenous cTnI was replaced with cTnI phosphorylated by protein kinase C-epsilon or mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used detergent-skinned mouse cardiac fiber bundles to measure changes in Ca(2+)-dependence of force. Compared with controls, fibers reconstituted with phosphorylated cTnI, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E were desensitized to Ca(2+), and maximum tension was as much as 27% lower, whereas fibers reconstituted with cTnI-T144E showed no change. In the in vitro motility assay actin filaments regulated by troponin complexes containing phosphorylated cTnI or cTnI-S43E/S45E/T144E showed both a decrease in Ca(2+) sensitivity and maximum sliding speed compared with controls, whereas filaments regulated by cTnI-S43E/S45E showed only decreased maximum sliding speed and filaments regulated by cTnI-T144E demonstrated only desensitization to Ca(2+). Our results demonstrate novel site specificity of effects of PKC phosphorylation on cTnI function and emphasize the complexity of modulation of the actin-myosin interaction by specific changes in the thin filament.

    Funded by: NHLBI NIH HHS: F32 HL 10409, P01 HL 62426, R01 HL 64035; NIAMS NIH HHS: R01 AR-30988; PHS HHS: T3207692

    The Journal of biological chemistry 2003;278;13;11265-72

  • Differentiation of nonbeating embryonic stem cells into beating cardiomyocytes is dependent on downregulation of PKC beta and zeta in concert with upregulation of PKC epsilon.

    Zhou X, Quann E and Gallicano GI

    Department of Cell Biology, Georgetown University Medical Center, Washington, DC 20007, USA.

    Cardiomyocyte differentiation overall has been analyzed in vivo and in vitro at the molecular level by homologous recombination, gene mutation studies, and by transgenics; however, the roles of many signal transduction mechanisms that drive this differentiation process are still not fully understood. One set of signal transduction components that has been studied in detail in mature, differentiated cardiomyocytes is the PKC isotype superfamily. However, while the function of each isotype is slowly being uncovered in adult cardiomyocytes, limited information persists concerning their function in the differentiation process of cardiomyocytes. To begin analyzing the function of specific PKC isotypes in the differentiation process, we employed an established model for differentiating ES cells into cardiomyocyte-positive embryoid bodies (EBs) in vitro. RT-PCR, Western analyses, and confocal microscopy all showed that the expression of specific PKC isotypes was significantly changed as ES cells differentiated into cardiomyocytes. More importantly, by using antagonists specific for each isotype we found that this change was a final step in the differentiation process. PKC beta and zeta downregulation served to promote differentiation (beating), while upregulation of PKC epsilon appeared to amplify differentiation (beating). Finally, melding classical tools (i.e., ionic exchange glass beads) with recently developed methods for differentiating ES cells creates a possible novel technique for investigating differentiation of ES cells into cardiomyocytes as well as other cell types.

    Developmental biology 2003;255;2;407-22

  • 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

  • A role for PKC-epsilon in Fc gammaR-mediated phagocytosis by RAW 264.7 cells.

    Larsen EC, Ueyama T, Brannock PM, Shirai Y, Saito N, Larsson C, Loegering D, Weber PB and Lennartz MR

    Centers for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA.

    Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC- epsilon in Fc gamma receptor (Fc gammaR)-dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG-opsonized beads. PKC- epsilon, but not PKC-delta, concentrated around the beads. PKC- epsilon accumulation was transient; apparent as a "flash" on target ingestion. Similarly, endogenous PKC- epsilon was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC- epsilon, but not PKC-alpha, PKC-delta, or PKC-gamma enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC- epsilon expressors was twice that of cells expressing GFP PKC-delta. Expression of the regulatory domain ( epsilon RD) and the first variable region ( epsilon V1) of PKC- epsilon inhibited uptake, whereas the corresponding PKC-delta region had no effect. Actin polymerization was enhanced on expression of GFP PKC- epsilon and epsilon RD, but decreased in cells expressing epsilon V1, suggesting that the epsilon RD and epsilon V1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC- epsilon in Fc gammaR-mediated phagocytosis that is independent of its effects on actin assembly.

    Funded by: NIGMS NIH HHS: GM50821

    The Journal of cell biology 2002;159;6;939-44

  • Conditional rescue of protein kinase C epsilon regulates ethanol preference and hypnotic sensitivity in adult mice.

    Choi DS, Wang D, Dadgar J, Chang WS and Messing RO

    Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California 94608, USA.

    Conventional gene targeting is a powerful tool to study the influence of specific genes on behavior. However, conclusions relevant for adult animals are limited by consequences of gene loss during development. Mice lacking protein kinase C epsilon (PKCepsilon) consume less alcohol and show greater acute sensitivity to alcohol than do wild-type mice. There are no selective inhibitors of PKCepsilon that can be administered systemically and cross the blood-brain barrier to test whether these phenotypes result from loss of PKCepsilon during development or in adulthood. Here we used conditional expression of PKCepsilon in the basal forebrain, amygdala, and cerebellum to rescue wild-type responses to alcohol in adult PKCepsilon(-/-) mice. Subsequent suppression of transgenic PKCepsilon restored PKCepsilon(-/-) behaviors. These findings establish that PKCepsilon signaling in the adult brain regulates alcohol consumption and sensitivity. If this extends to humans, then PKCepsilon inhibitors might prove useful as novel therapeutics for alcoholism.

    Funded by: NIAAA NIH HHS: AA08117

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;22;9905-11

  • Decreased anxiety-like behavior, reduced stress hormones, and neurosteroid supersensitivity in mice lacking protein kinase Cepsilon.

    Hodge CW, Raber J, McMahon T, Walter H, Sanchez-Perez AM, Olive MF, Mehmert K, Morrow AL and Messing RO

    Ernest Gallo Clinic and Research Center, Department of Neurology, University of California San Francisco, Emeryville, California 94608, USA.

    Mice lacking protein kinase Cepsilon (PKCepsilon) are supersensitive to positive allosteric modulators of gamma aminobutyrate type A (GABA(A)) receptors. Since many of these compounds are anxiolytic, we examined whether anxiety-like behavior is altered in these mice. PKCepsilon-null mice showed reduced anxiety-like behavior and reduced levels of the stress hormones corticosterone and adrenocorticotrophic hormone (ACTH). This was associated with increased sensitivity to neurosteroid modulators of GABA(A) receptors. Treatment of PKCepsilon-null mice with the GABA(A) receptor antagonist bicuculline restored corticosterone levels and anxiety-like behavior to wild-type levels. These results suggest that increased GABA(A) receptor sensitivity to neurosteroids contributes to reduced anxiety-like behavior and stress hormone responses in PKCepsilon-null mice. The findings also suggest PKCepsilon as a possible therapeutic target for development of anxiolytics.

    Funded by: NIA NIH HHS: AG-20079; NIAAA NIH HHS: AA-08117, AA-09981

    The Journal of clinical investigation 2002;110;7;1003-10

  • Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts.

    Saurin AT, Pennington DJ, Raat NJ, Latchman DS, Owen MJ and Marber MS

    Department of Cardiology, KCL, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK. adrian.saurin@kcl.ac.uk

    Objective: Activation of protein kinase C (PKC) isoforms is associated with the cardioprotective effect of early ischaemic preconditioning (IP). PKC consists of at least 10 different isoforms, encoded by separate genes, which mediate distinct physiological functions. Although the PKC-epsilon isoform has been implicated in preconditioning, uncertainty remains. We investigated whether preconditioning still occurs in a mouse line lacking cardiac PKC-epsilon protein due to a targeted disruption within the pkc-epsilon allele.

    Methods: The isolated buffer-perfused hearts from knockout mice lacking PKC-epsilon (-/-) and sibling heterozygous mice (+/-), with a normal PKC-epsilon complement, were preconditioned by 4 x 4 min ischaemia/6 min reperfusion. Hearts then underwent 45 min of global ischaemia followed by 1.5 h of reperfusion.

    Results: In PKC-epsilon (+/-) hearts ischaemic preconditioning reduced infarction volume as a percentage of myocardial volume (24.3+/-4.5 vs. 41.3+/-4.7%, P<0.001). In contrast, in PKC-epsilon (-/-) hearts preconditioning failed to diminish infarction (36.4+/-2.9 vs. 38.8+/-4.5%). Surprisingly however, although preconditioning did not reduce infarct size, it did enhance contractile recovery in PKC-epsilon (-/-) mice (43.1+/-3.9 vs. 24.9+/-5.1%, P<0.05), similar to the level seen in PKC-epsilon (+/-) hearts (35.2+/-3.9 vs. 20.9+/-5.0%, P<0.05).

    Conclusions: These data suggest that PKC-epsilon is essential for the reduction in infarction that follows early ischaemic preconditioning, but is not associated with the improvement in functional recovery.

    Cardiovascular research 2002;55;3;672-80

  • Activation and phosphatidylinositol 3-kinase-dependent phosphorylation of protein kinase C-epsilon by the B cell antigen receptor.

    Ting HC, Christian SL, Burgess AE and Gold MR

    Department of Microbiology and Immunology, University of British Columbia, 6174 University Blvd., Vancouver, BC, Canada V6T 1Z3.

    Protein kinase C (PKC) enzymes play an important role in B cell antigen receptor (BCR) signaling, linking the BCR to the activation of mitogen-activated protein kinases as well as the NF-kappa B, and AP-1 transcription factors. There are eleven different PKC isoforms, each of which is likely to have a unique set of substrates and hence a unique role in signal transduction. Although PKC-alpha, PKC-beta, PKC-delta, and PKC-zeta have been shown to be targets of BCR signaling, the full spectrum of PKC enzymes that are activated by the BCR remains to be determined. In this report, we show that PKC-epsilon is a target of BCR signaling. We found that PKC-epsilon is highly expressed in B cells and that BCR engagement causes PKC-epsilon to translocate from the cytosol to cellular membranes. This presumably reflects the binding of PKC-epsilon to its membrane-associated lipid activator, diacylglycerol. We also found that BCR engagement resulted in the phosphatidylinositol 3-kinase-dependent phosphorylation of PKC-epsilon. This modification may promote the full activation of PKC-epsilon. Activation of PKC-epsilon could be a key event in BCR signaling since PKC-epsilon has been strongly linked to cell survival and proliferation in other cell types.

    Immunology letters 2002;82;3;205-15

  • Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor.

    Saito Y, Hojo Y, Tanimoto T, Abe J and Berk BC

    Center for Cardiovascular Research, University of Rochester, Rochester, New York 14642, USA.

    Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.

    Funded by: NHLBI NIH HHS: HL49192, HL63462

    The Journal of biological chemistry 2002;277;26;23216-22

  • The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility.

    Besson A, Wilson TL and Yong VW

    Department of Oncology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

    Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrin-mediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKCepsilon positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKCepsilon activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin beta chain and activated PKCepsilon. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKCepsilon-induced adhesion and migration, suggesting that RACK1 links PKCepsilon to integrin beta chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility.

    The Journal of biological chemistry 2002;277;24;22073-84

  • Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKC epsilon knockout mouse hearts.

    Jin ZQ, Zhou HZ, Zhu P, Honbo N, Mochly-Rosen D, Messing RO, Goetzl EJ, Karliner JS and Gray MO

    Cardiology Section, Veterans Affairs Medical Center, San Francisco, California 94121, USA.

    Sphingosine-1-phosphate (S1P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S1P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of 10 nM S1P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with 10 nM of the ganglioside GM-1 also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-1 but not S1P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-1, which enhances endogenous S1P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S1P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.

    Funded by: NHLBI NIH HHS: R01 HL-31809, R01 HL-52141

    American journal of physiology. Heart and circulatory physiology 2002;282;6;H1970-7

  • Inhibition of glycogen synthesis by fatty acid in C(2)C(12) muscle cells is independent of PKC-alpha, -epsilon, and -theta.

    Cazzolli R, Craig DL, Biden TJ and Schmitz-Peiffer C

    Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.

    We have previously shown that glycogen synthesis is reduced in lipid-treated C(2)C(12) skeletal muscle myotubes and that this is independent of changes in glucose uptake. Here, we tested whether mitochondrial metabolism of these lipids is necessary for this inhibition and whether the activation of specific protein kinase C (PKC) isoforms is involved. C(2)C(12) myotubes were pretreated with fatty acids and subsequently stimulated with insulin for the determination of glycogen synthesis. The carnitine palmitoyltransferase-1 inhibitor etomoxir, an inhibitor of beta-oxidation of acyl-CoA, did not protect against the inhibition of glycogen synthesis caused by the unsaturated fatty acid oleate. In addition, although oleate caused translocation, indicating activation, of individual PKC isoforms, inhibition of PKC by pharmacological agents or adenovirus-mediated overexpression of dominant negative PKC-alpha, -epsilon, or -theta mutants was unable to prevent the inhibitory effects of oleate on glycogen synthesis. We conclude that neither mitochondrial lipid metabolism nor activation of PKC-alpha, -epsilon, or -theta plays a role in the direct inhibition of glycogen synthesis by unsaturated fatty acids.

    American journal of physiology. Endocrinology and metabolism 2002;282;6;E1204-13

  • Regulation of novel protein kinase C epsilon by phosphorylation.

    Cenni V, Döppler H, Sonnenburg ED, Maraldi N, Newton AC and Toker A

    Boston Biomedical Research Institute, Watertown, MA 02472, USA.

    The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKCepsilon isoform, these are Thr(566) in the activation loop, Thr(710) in the turn motif and Ser(729) in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKCepsilon kinase activity in controlling the phosphorylation of Thr(566) and Ser(729). In NIH 3T3 fibroblasts, PKCepsilon migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr(566) and Ser(729). Cells transfected with an active PDK-1 allele also resulted in increased PKCepsilon Thr(566) and Ser(729) phosphorylation, whereas an active myristoylated PKCepsilon mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKCepsilon were not phosphorylated at Ser(729) in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr(566), an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKCepsilon in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P(3), concomitant with an increase in Ser(729) phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

    Funded by: NCI NIH HHS: CA75134; NIGMS NIH HHS: GM43154

    The Biochemical journal 2002;363;Pt 3;537-45

  • Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon-MAPK interactions and differential MAPK activation in PKCepsilon-induced cardioprotection.

    Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, Bolli R and Ping P

    Department of Physiology, University of Louisville, and the Jewish Hospital Heart and Lung Institute, Louisville, KY 40202, USA.

    Although activation of protein kinase C (PKC) epsilon and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKCepsilon signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKCepsilon and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKCepsilon, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKCepsilon greatly increased mitochondrial PKCepsilon expression and activity, which was concomitant with increased mitochondrial interaction of PKCepsilon with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKCepsilon activity, as the interactions were also observed in mice expressing inactive PKCepsilon. However, although both active and inactive PKCepsilon bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKCepsilon but not in mice expressing inactive PKCepsilon. Examination of potential downstream targets of mitochondrial PKCepsilon-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKCepsilon forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKCepsilon-ERK modules appears to play a role in PKCepsilon-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.

    Funded by: NHLBI NIH HHS: HL-43151, HL-55757, HL-63901, HL-65431, HL-68088

    Circulation research 2002;90;4;390-7

  • Expression of activated PKC epsilon (PKC epsilon) protects the ischemic heart, without attenuating ischemic H(+) production.

    Cross HR, Murphy E, Bolli R, Ping P and Steenbergen C

    Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA. cross017@mc.duke.edu

    PKC epsilon is a PKC isoform that translocates during preconditioning and may mediate cardioprotection. To investigate whether PKC epsilon activation is cardioprotective, Langendorff-perfused hearts from wild-type (WT) mice and from mice expressing constitutively active mutant PKC epsilon were subjected to 20 min ischemia and 40 min reperfusion while(31)P NMR spectra were acquired. Pre-ischemic glycogen levels were similar in WT and PKC epsilon hearts. During ischemia, ATP fell less in PKC epsilon than in WT hearts. Ischemic intracellular pH, however, was similar in WT and PKC epsilon hearts. During reperfusion, recovery of contractile function and ATP were greater in PKC epsilon than WT hearts. In conclusion, expression of activated PKC epsilon protected hearts from post-ischemic energetic and contractile dysfunction, consistent with the proposed cardioprotective role of PKC epsilon. Protection occurred in the PKC epsilon hearts without attenuation of ischemic H(+) production, implying that, at least in this ischemic model, reduced acidification during ischemia is not necessary for cardioprotection.

    Funded by: NHLBI NIH HHS: R01 HL039752

    Journal of molecular and cellular cardiology 2002;34;3;361-7

  • Molecular conformation dictates signaling module formation: example of PKCepsilon and Src tyrosine kinase.

    Song C, Vondriska TM, Wang GW, Klein JB, Cao X, Zhang J, Kang YJ, D'Souza S and Ping P

    Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky 40202, USA.

    Our laboratory has conducted multiple functional proteomic analyses to characterize the components of protein kinase C (PKC)epsilon cardioprotective signaling complexes and found that activation of PKCepsilon induces dynamic modulation of these complexes. In addition, it is known that signal transduction within a complex involves the formation of modules, one of which has been shown to include PKCepsilon and Src tyrosine kinase in the rabbit heart. However, the cellular mechanisms that define the assembly of PKCepsilon modules remain largely unknown. To address this issue, the interactions between PKCepsilon and Src were studied. We used recombinant proteins of wild-type PKCepsilon (PKCepsilon-WT) and open conformation mutants of the kinase (PKCepsilon-AE5 and PKCepsilon-AN59), the regulatory and catalytic domains of PKCepsilon, along with glutathione-S-transferase (GST) fusion proteins of Src (GST-Src) and two domains of Src (GST-SH2 and GST-SH3). GST pulldown assays demonstrated that Src and PKCepsilon are binding partners and that the interaction between PKCepsilon and Src appears to involve multiple sites. This finding was supported for endogenous PKCepsilon and Src in the murine heart using immunofluorescence-based confocal microscopy and coimmunoprecipitation. Furthermore, PKCepsilon-WT and GST-Src interactions were significantly enhanced in the presence of phosphatidyl-L-serine, an activator of PKC, indicating that Src favors interaction with activated PKCepsilon. This finding was confirmed when the PKCepsilon-WT was replaced with PKCepsilon-AE5 or PKCepsilon-AN59, demonstrating that the conformation of PKCepsilon is a critical determinant of its interactions with Src. Together, these results illustrate that formation of a signaling module between PKCepsilon and Src involves specific domains within the two molecules and is governed by the molecular conformation of PKCepsilon.

    Funded by: NHLBI NIH HHS: HL-43721, HL-59225, HL-63760, HL-63901, HL-65431, HL-66358

    American journal of physiology. Heart and circulatory physiology 2002;282;3;H1166-71

  • Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection.

    Ping P, Song C, Zhang J, Guo Y, Cao X, Li RC, Wu W, Vondriska TM, Pass JM, Tang XL, Pierce WM and Bolli R

    Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky, USA. peipeiping@hotmail.com

    The epsilon isoform of protein kinase C (PKCepsilon) is a member of the PKC family of serine/threonine kinases and plays a critical role in protection against ischemic injury in multiple organs. Functional proteomic analyses of PKCepsilon signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCepsilon orchestrates cardioprotection are poorly understood. Here we report that Lck, a member of the Src family of tyrosine kinases, forms a functional signaling module with PKCepsilon. In cardiac cells, PKCepsilon interacts with, phosphorylates, and activates Lck. In vivo studies showed that cardioprotection elicited either by cardiac-specific transgenic activation of PKCepsilon or by ischemic preconditioning enhances the formation of PKCepsilon-Lck modules. Disruption of these modules, via ablation of the Lck gene, abrogated the infarct-sparing effects of these two forms of cardioprotection, indicating that the formation of PKCepsilon-Lck signaling modules is required for the manifestation of a cardioprotective phenotype. These findings demonstrate, for the first time to our knowledge, that the assembly of a module (PKCepsilon-Lck) is an obligatory step in the signal transduction that results in a specific phenotype. Thus, PKCepsilon-Lck modules may serve as novel therapeutic targets for the prevention of ischemic injury.

    Funded by: NCRR NIH HHS: 1S10RR11368-01A1; NHLBI NIH HHS: HL-63901, HL43151, HL55757, HL65431, HL68088, R01 HL055757, R01 HL063901, R01 HL065431, R01 HL068088, R37 HL055757, R37 HL063901

    The Journal of clinical investigation 2002;109;4;499-507

  • alpha 1 Connexin (connexin43) gap junctions and activities of cAMP-dependent protein kinase and protein kinase C in developing mouse heart.

    Duncan JC and Fletcher WH

    Department of Pathology and Human Anatomy, Division of Anatomy, Loma Linda University School of Medicine, Loma Linda, California, USA.

    alpha 1 Connexin (connexin43) is the dominant gap junction protein of the developing and mature heart where it forms channels that mediate intercellular electrical and metabolic coupling events that are critical for heart function. alpha1 connexin channels are rapidly and reversibly gated by actions of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), and disruption of consensus sites for these phosphorylations are associated with severe heart malformations. However, there have been no reports on the relative activities of PKA or PKC in early heart formation. Nor has the presence and phosphorylation state of alpha1 connexin been documented in these same developmental stages. To begin these studies, we used hearts from 8.5-18.5 dpc (days postcoitus) mouse embryos, postpartum pups, and adults. Membrane or supernatant fractions were used for immunoblots to assess the amounts and distribution of alpha1 connexin protein and each protein kinase. Phosphotransferase assays were done to document the endogenous activities of PKA and PKC. Three species of alpha1 connexin at 44, 46, and 49 kDa were evident in 8.5- and 9.5-dpc embryos and adult hearts, but the 49-kDa band was not consistently found in 10.5 dpc or embryos through 18.5 dpc, although it was robust in adult heart. The amount of PKA was minimal in 8.5-dpc hearts but rose thereafter and was maximal by 10.5 dpc and remained stable throughout development. Catalytic activity of this enzyme was minimal in 8.5-dpc hearts then rose thereafter and was maximal by 10.5 dpc of development. PKC delta was confined mainly to membrane fractions, whereas PKC epsilon had supernatant- and membrane-associated forms. Both enzyme isoforms showed large fluctuations throughout development. In 8.5- and 9.5-dpc hearts, PKC catalytic activity was maximal but, by 10.5 dpc, activity dramatically declined and remained low thereafter. The results demonstrate that alpha1 connexin is present at the heart tube stage (8.5 dpc) of development onward and provide evidence suggesting that channels formed by this protein are dynamically regulated by PKA and PKC, especially in 8.5- and 9.5-day embryonic hearts, which are crucial times for heart formation and left/right patterning in general.

    Funded by: NHLBI NIH HHS: HL53374

    Developmental dynamics : an official publication of the American Association of Anatomists 2002;223;1;96-107

  • Regulation of tumor invasion and metastasis in protein kinase C epsilon-transformed NIH3T3 fibroblasts.

    Tachado SD, Mayhew MW, Wescott GG, Foreman TL, Goodwin CD, McJilton MA and Terrian DM

    Department of Anatomy and Cell Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina 27858, USA.

    Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCepsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCepsilon contains an actin binding site that is essential for the formation of elongate invadopodial-like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCepsilon-transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCepsilon remained oncogenic and tumorigenic in this experimental system. We propose that PKCepsilon directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries.

    Funded by: NIEHS NIH HHS: ES8397

    Journal of cellular biochemistry 2002;85;4;785-97

  • Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection.

    Castrillo A, Pennington DJ, Otto F, Parker PJ, Owen MJ and Boscá L

    Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.

    To assess directly the role of protein kinase C (PKC)epsilon in the immune system, we generated mice that carried a homozygous disruption of the PKCepsilon locus. PKCepsilon(-/-) animals appeared normal and were generally healthy, although female mice frequently developed a bacterial infection of the uterus. Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta. Further analysis revealed that LPS-stimulated macrophages from PKCepsilon(-/-) mice were deficient in the induction of nitric oxide synthase (NOS)-2, demonstrating a decrease in the activation of IkappaB kinase, a reduction in IkappaB degradation, and a decrease in nuclear factor (NF)kappaB nuclear translocation. After intravenous administration of Gram-negative or Gram-positive bacteria, PKCepsilon(-/-) mice demonstrated a significantly decreased period of survival. This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages. Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

    The Journal of experimental medicine 2001;194;9;1231-42

  • Ethanol consumption and behavioral impulsivity are increased in protein kinase Cgamma null mutant mice.

    Bowers BJ and Wehner JM

    Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, USA. bbowers@colorado.edu

    Etiological factors influencing the development of alcoholism are complex and, at a minimum, include an interaction between polygenic factors and personality and biological traits. Human and animal studies suggest that some genes may regulate both the traits associated with alcohol abuse, such as decreased sensitivity or anxiety, and vulnerability to alcoholism. The identification of these genes could elucidate neurochemical pathways that are important in the development of alcohol abuse. Results from the present study indicate that the gene encoding the neuronal-specific gamma subtype of protein kinase C (PKCgamma) influences both ethanol consumption and behavioral impulsivity, a personality characteristic associated with Type II alcoholics, in a pleiotropic manner. Mice lacking PKCgamma consume more ethanol in a two-bottle choice paradigm and also demonstrate increased behavioral impulsivity in an appetitive-signaled nosepoke task when compared with wild-type littermate control mice. Therefore, PKCgamma may be an important mechanism within the cell that mediates one or more neurochemical pathways relevant to an increased predisposition to alcoholism.

    Funded by: NIAAA NIH HHS: AA-00141, AA-03527, AA-11275

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;21;RC180

  • Protein kinase C isoform expression and activity in the mouse heart.

    Schreiber KL, Paquet L, Allen BG and Rindt H

    Montreal Heart Institute, Research Center, Montreal, Quebec, Canada H1T 1C8.

    The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.

    Funded by: Canadian Institutes of Health Research: 14725

    American journal of physiology. Heart and circulatory physiology 2001;281;5;H2062-71

  • Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy.

    Aronow BJ, Toyokawa T, Canning A, Haghighi K, Delling U, Kranias E, Molkentin JD and Dorn GW

    Department of Developmental Biology, Children's Hospital Research Center, Cincinnati, OH 45229, USA.

    To define molecular mechanisms of cardiac hypertrophy, genes whose expression was perturbed by any of four different transgenic mouse hypertrophy models [protein kinase C-epsilon activation peptide (PsiepsilonRACK), calsequestrin (CSQ), calcineurin (CN), and Galpha(q)] were compared by DNA microarray analyses using the approximately 8,800 genes present on the Incyte mouse GEM1. The total numbers of regulated genes (tens to hundreds) correlated with phenotypic severity of the model (Galpha(q) > CN > CSQ > PsiepsilonRACK), but demonstrated that no single gene was consistently upregulated. Of the three models exhibiting pathological hypertrophy, only atrial natriuretic peptide was consistently upregulated, suggesting that transcriptional alterations are highly specific to individual genetic causes of hypertrophy. However, hierarchical-tree and K-means clustering analyses revealed that subsets of the upregulated genes did exhibit coordinate regulatory patterns that were unique or overlapping across the different hypertrophy models. One striking set consisted of apoptotic genes uniquely regulated in the apoptosis-prone Galpha(q) model. Thus, rather than identifying a single common hypertrophic cardiomyopathy gene program, these data suggest that extensive groups of genes may be useful for the prediction of specific underlying genetic determinants and condition-specific therapeutic approaches.

    Funded by: NHLBI NIH HHS: HL-26057, HL-52318, HL-58010, HL-64018, HL/HD-59888; NIEHS NIH HHS: ES-08822; OCPHP CDC HHS: P40-PR-13358

    Physiological genomics 2001;6;1;19-28

  • Protein kinase C-epsilon transgenic mice: a unique model for metastatic squamous cell carcinoma.

    Jansen AP, Verwiebe EG, Dreckschmidt NE, Wheeler DL, Oberley TD and Verma AK

    Department of Human Oncology, Medical School, University of Wisconsin, Madison, 53792, USA.

    Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most common forms of human skin cancer. BCC is slow growing and mostly localized, whereas SCC metastasizes to the regional lymph nodes and subsequently to distal organs. In murine skin carcinogenesis models for SCC, the incidence of metastasis is very low. We report here that FVB/N transgenic mice, which overexpress (approximately 18-fold) epitope-tagged protein kinase C-epsilon (T7-PKCepsilon) protein in the epidermis provide a unique murine model system for highly malignant/metastatic SCC. Skin tumors were developed by the initiation-promotion protocol (initiation with 100 nmol 7,12-dimethyl-benz[a]anthracene; promotion with 5 nmol 12-O-tetradecanoylphorbol-13-acetate twice weekly). T7-PKCepsilon transgenic mice showed 92% suppression of papilloma development compared with wild-type littermates after 23 weeks of tumor promotion. However, within 15-20 weeks of 12-O-tetradecanoylphorbol-13-acetate promotion, 40% of T7-PKCepsilon mice developed at least one carcinoma compared with 7% of the wild-type mice. All carcinomas from T7-PKCepsilon mice appeared without prior papilloma formation. Interestingly, 7,12-dimethyl-benz[a]anthracene alone resulted in the development of squamous cell carcinomas in 22% of T7-PKCepsilon mice, whereas wild-type littermates developed no tumors. Histopathological analysis of tumors from multiple T7-PKCepsilon mice revealed moderately differentiated SCC invading the dermal region with neoplasia appearing to originate and invade from the hair follicle. Carcinomas of T7-PKCepsilon mice rapidly metastasized to regional lymph nodes within 3 weeks of appearance. In wild-type mice, the grade of the invading tumors, originating from interfollicular epidermis, was pathologically categorized as well-differentiated SCC and remained localized to the dermis. The T7-PKCepsilon transgenic mice may provide a rapid and unique in vivo model to investigate metastatic SCC.

    Funded by: NCI NIH HHS: CA35368

    Cancer research 2001;61;3;808-12

  • Biochemical and morphogenic effects of the interaction between protein kinase C-epsilon and actin in vitro and in cultured NIH3T3 cells.

    Hernandez RM, Wescott GG, Mayhew MW, McJilton MA and Terrian DM

    Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858, USA.

    Protein kinase C-epsilon coordinately regulates changes in cell growth and shape. Cells overproducing protein kinase C-epsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions that are reminiscent of the morphology observed in ras-transformed fibroblasts. Here we report that the regulatory C1 domain contains an actin binding hexapeptide motif that is essential for the morphogenic effects of protein kinase C-epsilon in cultured NIH3T3 murine fibroblasts. The extension of elongate processes by protein kinase C-epsilon transformed fibroblasts appeared to be driven by a kinase-independent mechanism that required organized networks of both actin and microtubules. Flow cytometry of phalloidin-stained cells demonstrated that protein kinase C-epsilon significantly increased the cellular content of polymerized actin in NIH3T3 cells. Studies with a cell-free system suggest that protein kinase C-epsilon inhibits the in vitro disassembly of actin filaments, is capable of desequestering actin monomers from physiologically relevant concentrations of thymosin beta4, and increases the rate of actin filament elongation by decreasing the critical concentration of actin. Based on these and other observations, it is proposed that protein kinase C-epsilon may function as a terminal downstream effector in at least one of the signaling pathways that mitogens engage to initiate outgrowth of cellular protrusions.

    Funded by: NIEHS NIH HHS: ES8397

    Journal of cellular biochemistry 2001;83;4;532-46

  • Reduced ethanol withdrawal severity and altered withdrawal-induced c-fos expression in various brain regions of mice lacking protein kinase C-epsilon.

    Olive MF, Mehmert KK, Nannini MA, Camarini R, Messing RO and Hodge CW

    Department of Neurology and Ernest Gallo Clinic and Research Center, University of California at San Francisco, 5858 Horton Street, Emeryville, CA 94608, USA.

    Withdrawal from chronic ethanol consumption can be accompanied by motor seizures, which may be a result of altered GABA(A) receptor function. Recently, we have generated and characterized mice lacking the epsilon isoform of protein kinase C as being supersensitive to the behavioral and biochemical effects of positive GABA(A) receptor allosteric modulators, including ethanol. The aim of the present study was to determine whether protein kinase C-epsilon null mutant mice display altered seizure severity during alcohol withdrawal. In addition, we used c-fos immunohistochemistry immediately following seizure assessment to identify potential brain regions involved in any observed differences in withdrawal severity. Mice were allowed to consume an ethanol-containing or control liquid diet as the sole source of food for 14 days. During the 7-h period following removal of the diet, both ethanol-fed wild-type and protein kinase C-epsilon null mutant mice displayed an overall increase in Handling-Induced Convulsion score versus control-fed mice. However, at 6 and 7h following diet removal, the Handling-Induced Convulsion score was reduced in ethanol-fed protein kinase C-epsilon null mutant mice compared to ethanol-fed wild-type mice. Ethanol-fed protein kinase C-epsilon null mutant mice also exhibited a decrease in the number of Fos-positive cells in the lateral septum, and an increase in the number of Fos-positive cells in the dentate gyrus, mediodorsal thalamus, paraventricular nuclei of the thalamus and hypothalamus, and substantia nigra compared to ethanol-fed wild-type mice. These data demonstrate that deletion of protein kinase C-epsilon results in diminished progression of ethanol withdrawal-associated seizure severity, suggesting that selective pharmacological inhibitors of protein kinase C-epsilon may be useful in the treatment of seizures during alcohol withdrawal. These data also provide insight into potential brain regions involved in generation or suppression of ethanol withdrawal seizures.

    Funded by: NIAAA NIH HHS: AA08117, AA09981

    Neuroscience 2001;103;1;171-9

  • Reduced operant ethanol self-administration and in vivo mesolimbic dopamine responses to ethanol in PKCepsilon-deficient mice.

    Olive MF, Mehmert KK, Messing RO and Hodge CW

    Department of Neurology, Ernest Gallo Clinic & Research Center, University of California San Francisco, 5858 Horton Street, Suite 200, Emeryville, CA 94608, USA.

    There is increasing evidence that individual protein kinase C (PKC) isozymes mediate specific effects of ethanol on the nervous system. In addition, multiple lines of evidence suggest that the mesoaccumbens dopamine reward system is critically involved in the rewarding and reinforcing effects of ethanol. Yet little is known about the role of individual PKC isozymes in ethanol reinforcement processes or in regulation of mesolimbic systems. In this study, we report that mice lacking the epsilon isoform of PKC (PKCepsilon) show reduced operant ethanol self-administration and an absence of ethanol-induced increase in extracellular dopamine levels in the nucleus accumbens. PKCepsilon null mice exhibited a 53% decrease in alcohol-reinforced operant responses under basal conditions, as well as following ethanol deprivation. Behavioural analysis revealed that while both genotypes had the same number of drinking bouts following deprivation, PKCepsilon null mice demonstrated a 61% reduction in number of ethanol reinforcers per bout and a 57% reduction in ethanol-reinforced response rate. In vivo microdialysis experiments showed that, in contrast to wild-type mice, PKCepsilon null mice exhibited no change in extracellular levels of dopamine in the nucleus accumbens following acute administration of ethanol (1 and 2 g/kg i.p.), while mesolimbic dopamine responses to cocaine (20 mg/kg i.p.) or high potassium (100 mM) in these mice were comparable with that of wild-types. These data provide further evidence that increases in extracellular mesolimbic dopamine levels contribute to the reinforcing effects of ethanol, and indicate that pharmacological agents inhibiting PKCepsilon may be useful in the treatment of alcohol dependence.

    Funded by: NIAAA NIH HHS: AA09981

    The European journal of neuroscience 2000;12;11;4131-40

  • Transgenic mice overexpressing protein kinase C epsilon in their epidermis exhibit reduced papilloma burden but enhanced carcinoma formation after tumor promotion.

    Reddig PJ, Dreckschmidt NE, Zou J, Bourguignon SE, Oberley TD and Verma AK

    Department of Human Oncology, Medical School, University of Wisconsin, Madison 53792, USA.

    To determine the role that protein kinase C epsilon (PKCepsilon) may play in skin growth, differentiation, and tumor promotion, transgenic mice were generated that overexpressed an epitope-tagged protein kinase C epsilon (T7-PKCepsilon) in their epidermis using the human keratin 14 promoter. Three independent mouse lines that overexpressed the T7-PKCepsilon in their epidermis were produced. The three independent lines 206, 224, and 215 exhibited a 3-, 6-, and 18-fold elevation, respectively, in the level of PKCepsilon immunoreactive protein. Line 215 exhibited a 19-fold greater phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated kinase activity than line 224. Line 206 exhibited a low basal T7-PKCepsilon activity, which failed to be stimulated by phosphatidylserine and TPA. All of the line 215 transgenic mice (F0 to the F2 generation) displayed phenotypic changes in the skin. The phenotypic changes progressed gradually, starting around 4-5 months of age, with mild dryness of the tail accompanied by hair loss and inflammation at the base of the tail. Hyperproliferation and ulceration of the affected regions were observed around 7-8 months of age. The hyperproliferative epidermis from the affected regions exhibited an expansion of the suprabasal epidermal cells. Inflammation and/or ulceration were also observed in the dorsal skin, the ears, and around the eyes. The line 215 mice, which expressed the highest level of PKCepsilon, were evaluated for sensitivity to mouse skin tumor promotion by TPA. Tumors were elicited by the initiation (7,12-dimethylbenz[a]anthracene, 100 nmol)-promotion (TPA, 5 nmol/twice weekly) protocol. The papilloma burden was reduced by 95-96% for male and female T7-PKCepsilon mice compared to wild-type controls. However, carcinomas developed rapidly in the T7-PKCepsilon mice treated with 7, 12-dimethylbenz[a]anthracene and TPA. These carcinomas appeared to form independently of prior papilloma development. These results demonstrate that PKCepsilon is an important regulator of skin tumor development.

    Funded by: NCI NIH HHS: CA 35368

    Cancer research 2000;60;3;595-602

  • Expression and activity of protein kinase C isoenzymes during normal and abnormal murine palate development.

    Balasubramanian G, Amann JF and Reddy CS

    Department of Veterinary Biomedical Sciences, University of Missouri-Columbia, 65211, USA.

    Protein kinase C (PKC) plays a critical role in signal transduction, mediating various cellular events critical for normal development, including that of the palate. In vivo and in vitro studies suggest the relevance of the inhibition of PKC by the mycotoxin, secalonic acid D (SAD), to its induction of cleft palate (CP) in mice. In the present study, temporal and spatial expression and the activity of various PKC isoenzymes were studied in the control and SAD-exposed murine embryonic palate during gestational days (GD) 12-14.5 by western blotting, immunohistochemistry, and phosphotransfer assay. The Ca2+-dependent isoenzymes, PKC alpha and PKC betaII, showed significant expression on GD 12.0, which gradually decreased through GD 14.5, whereas PKC betaI and PKC gamma were negligible throughout. All Ca2+-independent isoenzymes (epsilon, delta, and zeta) were expressed more abundantly and, in contrast to the Ca2+-dependent ones, progressively increased with age. SAD failed to alter this pattern of expression but enhanced the phosphorylation of PKC epsilon throughout development. Immunohistochemical analysis revealed an isoenzyme-specific distribution of PKC between the epithelium and mesenchyme. As expected, SAD significantly inhibited the total Ca2+-dependent PKC activity in palatal extracts. Although total Ca2+-independent PKC activity in palatal extracts was unaffected by SAD, individual pure isoenzymes were either selectively inhibited (PKC zeta), stimulated (PKC delta), or unaffected (PKC epsilon) by SAD. These results show that PKC isoenzymes exhibit dynamic temporal and spatial patterns of expression and activity in the developing palate and that the induction of CP by SAD is associated with an alteration in their activation and/or activity.

    Funded by: NIDCR NIH HHS: DEOD 11822

    Journal of craniofacial genetics and developmental biology 2000;20;1;26-34

  • Supersensitivity to allosteric GABA(A) receptor modulators and alcohol in mice lacking PKCepsilon.

    Hodge CW, Mehmert KK, Kelley SP, McMahon T, Haywood A, Olive MF, Wang D, Sanchez-Perez AM and Messing RO

    Department of Neurology and Ernest Gallo Clinic and Research Center, University of California, San Francisco 94110, USA. chodge@itsa.ucsf. edu

    Several of the actions of ethanol are mediated by gamma-aminobutyrate type A (GABA(A)) receptors. Here we demonstrated that mutant mice lacking protein kinase C epsilon (PKCepsilon) were more sensitive than wild-type littermates to the acute behavioral effects of ethanol and other drugs that allosterically activate GABA(A) receptors. GABA(A) receptors in membranes isolated from the frontal cortex of PKCepsilon null mice were also supersensitive to allosteric activation by ethanol and flunitrazepam. In addition, these mutant mice showed markedly reduced ethanol self-administration. These findings indicate that inhibition of PKCepsilon increases sensitivity of GABA(A) receptors to ethanol and allosteric modulators. Pharmacological agents that inhibit PKCepsilon may be useful for treatment of alcoholism and may provide a non-sedating alternative for enhancing GABA(A) receptor function to treat other disorders such as anxiety and epilepsy.

    Nature neuroscience 1999;2;11;997-1002

  • A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice.

    Khasar SG, Lin YH, Martin A, Dadgar J, McMahon T, Wang D, Hundle B, Aley KO, Isenberg W, McCarter G, Green PG, Hodge CW, Levine JD and Messing RO

    Department of Internal Medicine and Oral Surgery, National Institutes of Health/University of California, USA.

    There is great interest in discovering new targets for pain therapy since current methods of analgesia are often only partially successful. Although protein kinase C (PKC) enhances nociceptor function, it is not known which PKC isozymes contribute. Here, we show that epinephrine-induced mechanical and thermal hyperalgesia and acetic acid-associated hyperalgesia are markedly attenuated in PKCepsilon mutant mice, but baseline nociceptive thresholds are normal. Moreover, epinephrine-, carrageenan-, and nerve growth factor- (NGF-) induced hyperalgesia in normal rats, and epinephrine-induced enhancement of tetrodotoxin-resistant Na+ current (TTX-R I(Na)) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCepsilon-selective inhibitor peptide. Our findings indicate that PKCepsilon regulates nociceptor function and suggest that PKCepsilon inhibitors could prove useful in the treatment of pain.

    Funded by: NIAAA NIH HHS: AA10036; NINDS NIH HHS: NS21647

    Neuron 1999;24;1;253-60

  • Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C.

    Zhou Q, Ruiz-Lozano P, Martone ME and Chen J

    Department of Medicine, UCSD-Salk Program in Molecular Medicine, University of California at San Diego, School of Medicine, La Jolla, California 92093-0613, USA.

    We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.

    Funded by: NCRR NIH HHS: RR04050

    The Journal of biological chemistry 1999;274;28;19807-13

  • Protein kinase C-theta is specifically localized on centrosomes and kinetochores in mitotic cells.

    Passalacqua M, Patrone M, Sparatore B, Melloni E and Pontremoli S

    Institute of Biological Chemistry, University of Genoa, Viale Benedetto XV, 1-16132 Genoa, Italy.

    In this study we provide evidence that the protein kinase C (PKC)-straight theta isoenzyme is recruited on to the mitotic spindle in dividing murine erythroleukaemia (MEL) cells and associates specifically with centrosome and kinetochore structures. None of the other PKC isoenzymes (-alpha, -delta, -epsilon, -mu and -zeta) expressed by MEL cells shows this localization on the mitotic spindle. An identical subcellular distribution of PKC-straight theta is also observed in dividing murine P3 myeloma cells and human LAN-5 neuroblastoma cells, indicating that this PKC isoenzyme interacts with the mitotic apparatus in mammalian cells. In phorbol-ester-treated non-growing MEL cells, a rapid change in the intracellular distribution of PKC-straight theta occurs. Under these conditions, PKC-straight theta is translocated from the nuclear to the cytosolic cell compartment, an event that is accompanied by phosphorylation of the PKC-straight theta molecule and is followed by its down-regulation. The recovery of cell growth capacity results in the concomitant reappearance of PKC-straight theta. Furthermore, when MEL cells acquire the differentiated non-growing phenotype, the level of PKC-straight theta is reduced to less than 5%, suggesting that this PKC isoenzyme is no longer required. We propose that, unlike other members of the PKC family, PKC-straight theta may play a role in cell proliferation.

    The Biochemical journal 1999;337 ( Pt 1);113-8

  • Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: transgenic rescue and interactions with gene background.

    McNamara RK, Stumpo DJ, Morel LM, Lewis MH, Wakeland EK, Blackshear PJ and Lenox RH

    Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. rkm@mail.med.upenn.edu

    The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an approximately 50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCepsilon expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCepsilon expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.

    Funded by: NIMH NIH HHS: R01 MH59959-01

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;24;14517-22

  • The catalytic domain of PKC-epsilon, in reciprocal PKC-delta and -epsilon chimeras, is responsible for conferring tumorgenicity to NIH3T3 cells, whereas both regulatory and catalytic domains of PKC-epsilon contribute to in vitro transformation.

    Wang QJ, Acs P, Goodnight J, Blumberg PM, Mischak H and Mushinski JF

    Laboratory of Genetics, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.

    Protein kinase C-epsilon (PKC-epsilon) has been shown to increase growth and cause malignant transformation when overexpressed in NIH3T3 cells, whereas PKC-delta reduced fibroblast growth. Two reciprocal chimeric proteins (PKC-epsilondelta and PKC-deltaepsilon were constructed by exchanging the regulatory and catalytic domains of PKC-delta and -epsilon and were stably overexpressed in NIH3T3 cells. Fibroblasts that overexpressed either chimera showed maximum cell density and morphology that were intermediate between cells overexpressing PKC-delta and those that overexpressed PKC-epsilon. Moreover, all lines that expressed chimeras were capable of anchorage-independent growth in the presence of TPA, which indicated that both the regulatory and catalytic domains of PKC-epsilon could independently induce NIH3T3 transformation, although the combination of both domains, as found in PKC-epsilon, was the most active form. In contrast, the translocation pattern and ability to induce tumors in nude mice was attributable to the catalytic domains exclusively. In particular, cells that expressed PKC-deltaepsilon retained PKC-epsilon's full potency of tumorgenicity when injected into nude mice. In sum, our findings not only reinforce the concept that only certain PKC isozymes contribute to carcinogenesis but also show that different domains of PKCs mediate the physiologically distinguishable events of transformation and tumorgenesis.

    Oncogene 1998;16;1;53-60

  • Up-regulation of protein kinase C-epsilon promotes the expression of cytokine-inducible nitric oxide synthase in RAW 264.7 cells.

    Díaz-Guerra MJ, Bodelón OG, Velasco M, Whelan R, Parker PJ and Boscá L

    Instituto de Bioquímica (Consejo Superior de Investigaciones Cientifícas), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.

    Stimulation of the murine macrophage RAW 264.7 cell line with phorbol esters fails to promote nitric oxide synthesis as occurs in rat hepatocytes or peritoneal macrophages. Transfection of RAW 264.7 cells with plasmids harboring protein kinase C (PKC) -epsilon isotype but not with PKC-alpha, -beta1, -delta, or constitutively active -alpha and -beta1 isotypes resulted in the expression of nitric oxide synthase type II (iNOS), as reflected by the synthesis of nitric oxide measured in the culture medium of transfected cells. cotransfection of RAW 264.7 cells with the -1592 to +121-base pair promoter region of the murine iNOS gene and PKC isotypes specifically induced the transactivation of this promoter in the case of the plasmids containing the PKC-epsilon isotype. The mechanism by which PKC-epsilon induced iNOS expression involved the activation of nuclear factor binding to kappaB sites (NF-kappaB) as deduced by the suppressive effect of pyrrolidine dithiocarbamate on nitric oxide synthesis, an inhibitor of NF-kappaB activation, and by the activation of kappaB sites in cells transfected with a vector containing a kappaB motif linked to a chloramphenicol acetyltransferase reporter gene. These results suggest that PKC-epsilon can regulate a pathway that promotes iNOS expression in macrophages in response to phorbol ester activation.

    The Journal of biological chemistry 1996;271;50;32028-33

  • The inhibition of differentiation caused by TGFbeta in fetal myoblasts is dependent upon selective expression of PKCtheta: a possible molecular basis for myoblast diversification during limb histogenesis.

    Zappelli F, Willems D, Osada S, Ohno S, Wetsel WC, Molinaro M, Cossu G and Bouché M

    Institute of Histology and General Embryology, University of Rome La Sapienza, Italy.

    Embryonic and fetal skeletal myoblasts are responsible for the formation of primary and secondary fibers in mammals, but the mechanism which diversifies their fate is unknown. In vitro, embryonic myoblasts are resistant to the differentiation inhibitory effects of transforming growth factor beta and phorbol esters. Thus, differential expression of specific molecules involved in the transduction of extracellular signals may contribute to the different phenotypes. We report here that protein kinase C theta, but none of the other known protein kinase C isoforms, is selectively expressed in fetal and postnatal muscle cells (at both the myoblast and myotube stage) in vitro and in vivo. By contrast, embryonic myoblasts and myotubes do not express protein kinase C theta in vitro or in vivo. This difference is causally related to a differential response to transforming growth factor beta, since overexpression of protein kinase C theta, but not of protein kinase C alpha or zeta, in embryonic myoblasts makes these cells sensitive to transforming growth factor beta. These data demonstrate for the first time that a protein kinase C isoform is a key component of the signal transduction cascade which follows exposure of myoblasts to transforming growth factor beta. They also suggest a specific role for protein kinase C theta in determining the fate of different myoblasts during muscle histogenesis.

    Funded by: Telethon: 245

    Developmental biology 1996;180;1;156-64

  • Developmental expression of MARCKS and protein kinase C in mice in relation to the exencephaly resulting from MARCKS deficiency.

    Blackshear PJ, Lai WS, Tuttle JS, Stumpo DJ, Kennington E, Nairn AC and Sulik KK

    Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, NC 27710, USA.

    The roles of protein kinase C and its substrates in development are poorly understood. Recently, we disrupted the mouse gene for a major cellular substrate for protein kinase C, the MARCKS protein (Proc. Natl. Acad. Sci. USA, 92, 944-948, 1995). The resulting phenotype consisted of universal perinatal lethality, agenesis of the corpus callosum and other forebrain commissures, and neuronal ectopia and other cortical and retinal lamination disturbances. These mice also had high frequencies of exencephaly (25% overall, 35% in females). In the present study, we have examined the normal expression of MARCKS and the various isozymes of protein kinase C at the time of cranial neural tube closure, in an attempt to correlate MARCKS expression in time and anatomical location with the exencephaly characteristic of MARCKS deficiency. Failure of neural tube closure occurred at various sites in the cranial neural tube, suggesting a cellular functional defect that was not limited to a specific location. Non-exencephalic MARCKS-deficient embryos appeared to be anatomically normal on embryonic day (E) 8.5-9.5. MARCKS and PKC alpha were expressed at the plasma membrane of the neuroepithelial cells comprising the future neural tube, as well as in the surface ectoderm and underlying mesenchyme. Endogenous protein kinase C species, comprising either or both alpha and delta, were capable of phosphorylating MARCKS in intact E8.5 embryos. Thus, MARCKS is expressed at the plasma membranes of the specific cell types involved in cranial neurulation; its deficiency presumably results in a still-to-be-elucidated functional defect in these cells that leads to exencephaly in a high proportion of cases.

    Funded by: NIAAA NIH HHS: AA08204; NIDDK NIH HHS: T32-DK-07012

    Brain research. Developmental brain research 1996;96;1-2;62-75

  • Protein kinase C isoforms epsilon, eta, delta and zeta in murine adipocytes: expression, subcellular localization and tissue-specific regulation in insulin-resistant states.

    Frevert EU and Kahn BB

    Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, MA 02215, USA.

    The Ca(2+)-insensitive protein kinase C (PKC) isoforms epsilon, eta, delta and zeta are possible direct downstream targets of phosphatidylinositol 3-kinase (P13-K), and might therefore be involved in insulin signalling. Although isoform-specific changes in PKC expression have been reported for skeletal muscle and liver in insulin-resistant states, little is known about these isoforms in adipocytes. Therefore we studied (1) expression and subcellular localization of these isoforms in murine adipocytes, (2) translocation of specific isoforms to membranes in response to treatment with insulin and phorbol 12-myristate 13-acetate (PMA) and (3) regulation of expression in insulin-resistant states. The PKC isoforms epsilon, eta, delta and zeta are expressed in adipocytes. Immunoreactivity for all isoforms is higher in the membranes than in the cytosol, but subcellular fractionation by differential centrifugation shows an isoform-specific distribution within the membrane fractions. PMA treatment of adipocytes induces translocation of PKC-epsilon and -delta from the cytosol to the membrane fractions. Insulin treatment does not alter the subcellular distribution of any of the isoforms. 3T3-L1 adipocytes express PKC-epsilon and -zeta, and PKC-epsilon expression increases with differentiation from preadipocytes to adipocytes. PKC-epsilon expression decreases in an adipose-specific and age/obesity-dependent manner in two insulin-resistant models, the brown-adipose-tissue-deficient mouse and db/db mouse compared with control mice. We conclude that, although none of the isoforms investigated seems to be activated by insulin, the decrease in PKC-epsilon expression might contribute to metabolic alterations in adipocytes associated with insulin resistance and obesity.

    Funded by: NIDDK NIH HHS: P30DK46200, R01 DK043051, R01DK43051, R01DK46930

    The Biochemical journal 1996;316 ( Pt 3);865-71

  • Functional expression and characterization of the mouse epitope tag-protein kinase C isoforms, alpha, beta I, beta II, gamma, delta and epsilon.

    Tseng CP and Verma AK

    Department of Human Oncology, Medical School, University of Wisconsin-Madison 53792, USA. ctseng@students.wisc.edu

    To simplify the detection of the exogenously produced protein kinase C (PKC) isoforms, we constructed the T7-Tag PKC alpha, betaI, betaII, gamma, delta and epsilon expression plasmids. T7-Tag sequences (AlaSerMetThrGlyGlyGlnGlnMetGlyArg) were inserted at the 5'-end of the translational initiation site. Transient transfection of T7-Tag PKC expression plasmids into CV-1 cells increased the levels of PKC protein, and PKC activity. T7-Tag-PKC epsilon, like native PKC epsilon, transactivated the transcription of a NF-kappa B reporter construct. These results indicate that the plasmids encoding T7-Tag PKC are functional and may be useful to define the role PKC isoforms play in cell proliferation, differentiation and tumor promotion.

    Gene 1996;169;2;287-8

  • Distribution of the extended family of protein kinase C isoenzymes in fetal organs of mice: an immunohistochemical study.

    Bareggi R, Grill V, Zweyer M, Narducci P and Martelli AM

    Dipartimento di Morfologia Umana Normale, Università degli Studi di Trieste, Italy.

    Using isoenzyme-specific antisera, we have studied the distribution of protein kinase C isoforms in fetal mouse organs at the developmental age of 17 days. Two different sets of antibodies, produced by different manufacturers, were employed in this study. The specificity of the antisera was tested by immunoblotting experiments using whole fetal mouse extracts. Immunohistochemistry was carried out by means of an alkaline phosphatase-conjugated secondary antibody. Analysis of fetal mouse longitudinal cryostat sections stained with the antibodies demonstrated a distinct distribution of protein kinase C isoforms in the tissues. Protein kinase C-alpha and C-beta I were present in all tissues examined, whereas the C-beta II isoform was absent in the lung and the liver. Protein kinase C-gamma was identified in brain, spinal ganglia, and adrenal gland. The C-epsilon isoenzyme was abundantly expressed in spinal ganglia and in the smooth muscle cells of the bronchial wall. Antisera to C-zeta and C-eta isoforms heavily stained liver, kidney, and spinal ganglia, whereas the C-theta isozyme was mainly detected in brain, stomach and kidney. Thus, protein kinase C-alpha, C-beta I, C-beta II, C-zeta, C-eta and C-theta were the isoforms present in many of the organs investigated. The two sets of antibodies gave slightly different results that might be ascribed to the different epitopes recognized by the antisera. One set of antisera was employed to investigate the distribution of the isoforms in selected organs from an earlier developmental age (15 days) and from adult animals.(ABSTRACT TRUNCATED AT 250 WORDS)

    Cell and tissue research 1995;280;3;617-25

  • Protein kinase C isoenzymes in mouse harderian gland. Differential expression of the alpha- and epsilon-isoforms during pregnancy. Protein kinase C-OC.

    Grill V, Martelli AM, Bareggi R, Santi S, Basa M, Zweyer M, Cocco L and Narducci P

    Dipartimento di Morfologia Umana Normale, Università di Trieste, Italy.

    Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-alpha, -epsilon, -zeta and -eta. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the alpha- and epsilon-isoforms. The 80-kDa native from of PKC-alpha almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C-epsilon appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant in the Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions.(ABSTRACT TRUNCATED AT 250 WORDS)

    Histochemistry and cell biology 1995;103;4;255-62

  • An immunohistochemical study of protein kinase C distribution in fetal mouse vertebral column.

    Bareggi R, Neri LM, Grill V, Cocco L and Martelli AM

    Dipartimento di Morfologia Umana Normale, Trieste, Italy.

    Using polyclonal antibodies we have studied the distribution of protein kinase C in fetal mouse low thoracic vertebrae. By means of a pan protein kinase C antiserum recognizing the catalytic domain of the enzyme, we show that protein kinase C is markedly expressed in chondrocytes before birth. The enzyme seems to be very abundant in the more mature cells that are close to ossification centres as well as the periphery of the intervertebral disc, although it can also be detected in chondrocytes. In order to establish which protein kinase C isoenzyme(s) the chondrocytes produce, we employed polyclonal isoenzyme-specific antisera developed against three calcium-dependent isoforms (alpha, beta, gamma) and three calcium-independent isoforms (delta, epsilon, zeta). Secondary antibody conjugated to alkaline phosphatase revealed that chondrocytes markedly express the beta-isoform. Cells were also weakly stained by the anti-epsilon serum. The immunostaining was completely abolished by pre-incubating primary antibodies with the peptide antigens to which they were raised. These results suggest that protein kinase C (and particularly the beta isoform) could play an important role in mouse fetal chondrogenesis of the vertebral column.

    Anatomy and embryology 1994;190;1;47-54

  • A new member of the third class in the protein kinase C family, PKC lambda, expressed dominantly in an undifferentiated mouse embryonal carcinoma cell line and also in many tissues and cells.

    Akimoto K, Mizuno K, Osada S, Hirai S, Tanuma S, Suzuki K and Ohno S

    Department of Molecular Biology, Yokohama City University School of Medicine, Japan.

    Protein kinase C (PKC)-related cDNA clones isolated from cDNA libraries of mouse P19 embryonal carcinoma cells and mouse brain encoded a 67-kDa protein, PKC lambda. PKC lambda shows the highest amino acid sequence identity with PKC zeta (72%), the third class of the PKC family. Northern blot analysis showed that the mRNA for PKC lambda is expressed in a wide variety of cells and tissues, including P19 and NIH 3T3 cells, as well as brain, kidney, testis, and ovary. In undifferentiated P19 cells, the mRNA for PKC lambda is the most abundant among all the PKC family members. The differentiation of P19 cells results in an increase in PKC alpha and epsilon, and a decrease in PKC lambda. Antiserum raised against a peptide of PKC lambda identified a 74-kDa protein in P19 cell extracts as well as in extracts from COS cells transfected with the PKC lambda expression plasmid. Autophosphorylation of the PKC lambda that immunoprecipitated with the specific antiserum was observed, indicating that PKC lambda possesses protein kinase activity. A phorbol ester binding assay using intact COS cells expressing PKC lambda failed to detect binding activity specific to PKC lambda at phorbol dibutylate concentrations up to 300 nM, suggesting that PKC lambda does not possess phorbol ester binding activity. These results, in conjunction with the results obtained in parallel experiments with PKC zeta and other PKC members, suggest a biochemical similarity between PKC lambda and zeta and their clear difference from other PKC members.

    The Journal of biological chemistry 1994;269;17;12677-83

  • Overexpressed protein kinase C-delta and -epsilon subtypes in NIH 3T3 cells exhibit differential subcellular localization and differential regulation of sodium-dependent phosphate uptake.

    Lehel C, Olah Z, Mischak H, Mushinski JF and Anderson WB

    Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.

    To examine the biological properties of protein kinase C (PKC)-delta and -epsilon NIH 3T3 cells were stably transfected with metallothionein-based expression vectors that overexpressed these isoforms. In addition to their inducibility by Zn2+, the protein levels of these two PKC subtypes, but not that of endogenous PKC-alpha, increased with increasing cell density. An unexpected role for Mg2+ in the subcellular localization of PKC-delta was found. This isoenzyme was predominantly membrane-associated when cell fractionation was carried out in the absence of Mg2+ but cytosolic when the fractionation was performed in the presence of 10 mM Mg2+. In contrast, the predominant localization of cytosolic PKC-alpha and of membrane-associated PKC-epsilon was not influenced by Mg2+. In vivo and in vitro studies of [3H]phorbol 12,13-dibutyrate binding in the overexpressing cell lines confirmed the cytosolic localization of PKC-alpha, the membrane-associated state of PKC-epsilon, and the presence of PKC-delta at both locations. Readdition of serum for 5 min to serum-starved, quiescent cell lines initiated the redistribution of PKC-alpha to the particulate fraction, while the location of PKC-delta and PKC-epsilon was not affected. Zn(2+)-induced overexpression of PKC-delta- and PKC-epsilon-stimulated sodium-dependent phosphate uptake. Overexpression of PKC-delta caused an increase in the Vmax of Na+/P(i) uptake, while overexpression of PKC-epsilon resulted in a decrease in Km for orthophosphate. A further stimulation of Na+/P(i) uptake in the overexpressing cells could be achieved by phorbol ester activation of endogenous PKC-alpha. These results suggest that each of the three PKC isotypes contribute to the regulation of sodium-dependent phosphate uptake, but through distinct mechanisms.

    The Journal of biological chemistry 1994;269;7;4761-6

  • A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin.

    Osada S, Mizuno K, Saido TC, Akita Y, Suzuki K, Kuroki T and Ohno S

    Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.

    Protein kinase C (PKC)-related cDNA clones isolated from mouse epidermis cDNA library encoded a 78-kDa protein, nPKC eta. nPKC eta contains a characteristic cysteine-rich repeat sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are conserved among PKC family members. However, nPKC eta lacks a putative Ca2+ binding region (C2 region) that is seen in conventional PKCs (alpha, beta I, beta II, gamma), but not in novel PKCs (nPKC delta, -epsilon, -zeta). nPKC eta shows the highest sequence similarity to nPKC epsilon (59.4% identity). The similarity extends to the NH2-terminal sequence (E region) which corresponds to one of the divergent regions (D1 region). Northern blot analysis showed that the mRNA for nPKC eta is highly expressed in the lung and skin but, in contrast to other members of the PKC family, only slightly expressed in the brain. nPKC eta expressed in COS cells shows phorbol ester binding activity with a similar affinity to nPKC epsilon. Antiserum raised against a COOH-terminal peptide of nPKC eta identified an 82-kDa protein in mouse lung extract as well as in an extract from COS cells transfected with the nPKC eta-cDNA expression plasmid. Autophosphorylation of nPKC eta immunoprecipitated with the specific antiserum was observed, indicating that nPKC eta is a protein kinase. These results clearly demonstrate the existence and the possible importance of nPKC eta as a member of the phorbol ester receptor/protein kinase, PKC, family.

    The Journal of biological chemistry 1990;265;36;22434-40

  • Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a.

    Wada H, Ohno S, Kubo K, Taya C, Tsuji S, Yonehara S and Suzuki K

    Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.

    By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.

    Biochemical and biophysical research communications 1989;165;1;533-8

  • Unique substrate specificity and regulatory properties of PKC-epsilon: a rationale for diversity.

    Schaap D, Parker PJ, Bristol A, Kriz R and Knopf J

    Ludwig Institute for Cancer Research, London, England.

    PKC-epsilon was isolated from a murine brain cDNA library. The clone, lambda 61PKC-epsilon, encoded a polypeptide of 737 amino acids that is homologous to other PKCs. Northern analysis showed that the 7 kb mRNA for this cDNA is widely expressed. The protein when expressed in COS-1 cells displayed phorbol ester-binding activity. However in order to detect the kinase activity of PKC-epsilon, it was necessary to employ a synthetic peptide substrate based upon the pseudosubstrate site. Subsequent analysis demonstrated that PKC-epsilon, while showing certain properties characteristic of the PKC family, has a quite distinct substrate specificity and is independent of Ca2+.

    FEBS letters 1989;243;2;351-7

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000007 G2C Mus musculus Mouse NRC Mouse NRC adapted from Collins et al (2006) 186
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000021 G2C Mus musculus Pocklington M3 Cluster 3 (mouse) from Pocklington et al (2006) 30
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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