G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
protein kinase, cAMP-dependent, regulatory, type II, alpha
G00000174 (Mus musculus)

Databases (8)

ENSG00000114302 (Ensembl human gene)
5576 (Entrez Gene)
320 (G2Cdb plasticity & disease)
PRKAR2A (GeneCards)
176910 (OMIM)
Marker Symbol
HGNC:9391 (HGNC)
Protein Expression
5023 (human protein atlas)
Protein Sequence
P13861 (UniProt)

Literature (85)

Pubmed - other

  • Identification of neuroglycan C and interacting partners as potential susceptibility genes for schizophrenia in a Southern Chinese population.

    So HC, Fong PY, Chen RY, Hui TC, Ng MY, Cherny SS, Mak WW, Cheung EF, Chan RC, Chen EY, Li T and Sham PC

    Department of Psychiatry, University of Hong Kong, Hong Kong SAR, China.

    Chromosome 3p was reported by previous studies as one of the regions showing strong evidence of linkage with schizophrenia. We performed a fine-mapping association study of a 6-Mb high-LD and gene-rich region on 3p in a Southern Chinese sample of 489 schizophrenia patients and 519 controls to search for susceptibility genes. In the initial screen, 4 SNPs out of the 144 tag SNPs genotyped were nominally significant (P < 0.05). One of the most significant SNPs (rs3732530, P = 0.0048) was a non-synonymous SNP in the neuroglycan C (NGC, also known as CSPG5) gene, which belongs to the neuregulin family. The gene prioritization program Endeavor ranked NGC 8th out of the 129 genes in the 6-Mb region and the highest among the genes within the same LD block. Further genotyping of NGC revealed 3 more SNPs to be nominally associated with schizophrenia. Three other genes (NRG1, ErbB3, ErbB4) involved in the neuregulin pathways were subsequently genotyped. Interaction analysis by multifactor dimensionality reduction (MDR) revealed a significant two-SNP interaction between NGC and NRG1 (P = 0.015) and three-SNP interactions between NRG1 and ErbB4 (P = 0.009). The gene NGC is exclusively expressed in the brain. It is implicated in neurodevelopment in rats and was previously shown to promote neurite outgrowth. Methamphetamine, a drug that may induce psychotic symptoms, was reported to alter the expression of NGC. Taken together, these results suggest that NGC may be a novel candidate gene, and neuregulin signaling pathways may play an important role in schizophrenia.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2010;153B;1;103-13

  • Bacillus anthracis edema toxin suppresses human macrophage phagocytosis and cytoskeletal remodeling via the protein kinase A and exchange protein activated by cyclic AMP pathways.

    Yeager LA, Chopra AK and Peterson JW

    Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-0610, USA. layeager@utmb.edu

    Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. It produces edema toxin (EdTx), a powerful adenylate cyclase that increases cyclic AMP (cAMP) levels in host cells. Because other cAMP-increasing agents inhibit key macrophage (MPhi) functions, such as phagocytosis, it was hypothesized that EdTx would exhibit similar suppressive activities. Our previous GeneChip data showed that EdTx downregulated MPhi genes involved in actin cytoskeleton remodeling, including protein kinase A (PKA). To further examine the role of EdTx during anthrax pathogenesis, we explored the hypothesis that EdTx treatment leads to deregulation of the cAMP-dependent PKA system, resulting in impaired cytoskeletal functions essential for MPhi activity. Our data revealed that EdTx significantly suppressed human MPhi phagocytosis of Ames spores. Cytoskeletal changes, such as decreased cell spreading and lowered F-actin content, were also observed for toxin-treated MPhis. Further, EdTx altered the protein levels and activity of PKA and exchange protein activated by cAMP (Epac), a recently identified cAMP-binding molecule. By using PKA- and Epac-selective cAMP analogs, we confirmed the involvement of both pathways in the inhibition of MPhi functions elicited by EdTx-generated cAMP. These results suggested that EdTx weakened the host immune response by increasing cAMP levels, which then signaled via PKA and Epac to cripple MPhi phagocytosis and interfered with cytoskeletal remodeling.

    Funded by: NIAID NIH HHS: N01-AI-30065, N01AI30065

    Infection and immunity 2009;77;6;2530-43

  • Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells.

    Martin BR, Deerinck TJ, Ellisman MH, Taylor SS and Tsien RY

    Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

    The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.

    Funded by: NCRR NIH HHS: P41 RR004050; NIDDK NIH HHS: DK54441; NIGMS NIH HHS: GM72033; NINDS NIH HHS: NS27177

    Chemistry & biology 2007;14;9;1031-42

  • Anchoring of protein kinase A-regulatory subunit IIalpha to subapically positioned centrosomes mediates apical bile canalicular lumen development in response to oncostatin M but not cAMP.

    Wojtal KA, Hoekstra D and van Ijzendoorn SC

    Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

    Oncostatin M and cAMP signaling stimulate apical surface-directed membrane trafficking and apical lumen development in hepatocytes, both in a protein kinase A (PKA)-dependent manner. Here, we show that oncostatin M, but not cAMP, promotes the A-kinase anchoring protein (AKAP)-dependent anchoring of the PKA regulatory subunit (R)IIalpha to subapical centrosomes and that this requires extracellular signal-regulated kinase 2 activation. Stable expression of the RII-displacing peptide AKAP-IS, but not a scrambled peptide, inhibits the association of RIIalpha with centrosomal AKAPs and results in the repositioning of the centrosome from a subapical to a perinuclear location. Concomitantly, common endosomes, but not apical recycling endosomes, are repositioned from a subapical to a perinuclear location, without significant effects on constitutive or oncostatin M-stimulated basolateral-to-apical transcytosis. Importantly, however, the expression of the AKAP-IS peptide completely blocks oncostatin M-, but not cAMP-stimulated apical lumen development. Together, the data suggest that centrosomal anchoring of RIIalpha and the interrelated subapical positioning of these centrosomes is required for oncostatin M-, but not cAMP-mediated, bile canalicular lumen development in a manner that is uncoupled from oncostatin M-stimulated apical lumen-directed membrane trafficking. The results also imply that multiple PKA-mediated signaling pathways control apical lumen development and that subapical centrosome positioning is important in some of these pathways.

    Molecular biology of the cell 2007;18;7;2745-54

  • Will diverse Tat interactions lead to novel antiretroviral drug targets?

    Harrich D, McMillan N, Munoz L, Apolloni A and Meredith L

    HIV-1 Molecular Virology Laboratory, Division of Immunology and Infectious Diseases, Queensland Institute of Medical Research, Royal Brisbane Hospital Post Office, Brisbane 4029, Qld, Australia. davidH@qimr.edu.au

    More than fifteen years following the description of Tat as a critical HIV gene expression regulatory protein, additional roles for Tat in HIV replication have been described, including reverse transcription. Tat achieves function through direct interaction with viral proteins, including reverse transcriptase, and numerous cellular proteins including cyclin T1, RNA polymerase II, protein kinase R (PKR), p300/CBP, and P/CAF. Despite our advanced knowledge of how Tat operates, this has not yet resulted in the discovery of effective agents capable of targeting various Tat functions. Nevertheless, Tat remains an attractive, virus-specific molecule and detailed understanding of specific protein interaction holds promise for future drug discovery.

    Current drug targets 2006;7;12;1595-606

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Molecular basis of AKAP specificity for PKA regulatory subunits.

    Gold MG, Lygren B, Dokurno P, Hoshi N, McConnachie G, Taskén K, Carlson CR, Scott JD and Barford D

    Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom.

    Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

    Funded by: NIDDK NIH HHS: DK54441

    Molecular cell 2006;24;3;383-95

  • Efficient trafficking of MDR1/P-glycoprotein to apical canalicular plasma membranes in HepG2 cells requires PKA-RIIalpha anchoring and glucosylceramide.

    Wojtal KA, de Vries E, Hoekstra D and van Ijzendoorn SC

    Section of Membrane Cell Biology, Department of Cell Biology, University Medical Center Groningen, University of Groningen, 9713 AV Groningen, The Netherlands.

    In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIalpha from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5'NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5'NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIalpha displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIalpha anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1.

    Molecular biology of the cell 2006;17;8;3638-50

  • Dynamic binding of PKA regulatory subunit RI alpha.

    Gullingsrud J, Kim C, Taylor SS and McCammon JA

    Department of Chemistry and Biochemistry, University of California, San Diego, California 92093, USA. jgulling@mccammon.ucsd.edu

    Recent crystal structures have revealed that regulatory subunit RIalpha of PKA undergoes a dramatic conformational change upon complex formation with the catalytic subunit. Molecular dynamics studies were initiated to elucidate the contributions of intrinsic conformational flexibility and interactions with the catalytic subunit in formation and stabilization of the complex. Simulations of a single RIalpha nucleotide binding domain (NBD), missing cAMP, showed that its C helix spontaneously occupies two distinct conformations: either packed against the nucleotide binding domain as in its cAMP bound structure, or extended into an intermediate form resembling that of the holoenzyme structure. C helix extension was not seen in a simulation of either RIalpha NBD. In a model complex containing both NBDs and the catalytic subunit, well-conserved residues at the interface between the NBDs in the cAMP bound form were found to stabilize the complex through contacts with the catalytic subunit. The model structure is consistent with available experimental data.

    Funded by: NIDDK NIH HHS: 1H DK07233; NIGMS NIH HHS: GM31749, GM34921

    Structure (London, England : 1993) 2006;14;1;141-9

  • Phosphoproteomic analysis of the human pituitary.

    Beranova-Giorgianni S, Zhao Y, Desiderio DM and Giorgianni F

    Charles B. Stout Neuroscience Mass Spectrometry Laboratory, The University of Tennessee Health Science Center, Memphis, TN 38163, USA.

    The pituitary is the central endocrine gland that regulates the functions of various target organs in the human body. Because of the pivotal regulatory role of the pituitary, it is essential to define on a global scale the components of the pituitary protein machinery, including a comprehensive characterization of the post-translational modifications of the pituitary proteins. Of particular interest is the examination of the phosphorylation status of the pituitary in health and disease. Towards the goal of global profiling of pituitary protein phosphorylation, we report here the application of the in-gel IEF-LC-MS/MS approach to the study of the pituitary phosphoproteome. The analytical strategy combined isoelectric focusing in immobilized pH gradient strips with immobilized metal ion affinity chromatography and mass spectrometry. With this method, a total of 50 phosphorylation sites were characterized in 26 proteins. Because the investigation involved primary tissue, the findings provide a direct glimpse into the phosphoprotein machinery operating within the human pituitary tissue microenvironment.

    Funded by: NINDS NIH HHS: NS 42843

    Pituitary 2006;9;2;109-20

  • Tumor reversion: protein kinase A isozyme switching.

    Cho-Chung YS and Nesterova MV

    Cellular Biochemistry Section, Basic Research Laboratory, National Cancer Institute, Building 10, Room 5B05, 9000 Rockville Pike, Bethesda, MD 20892-1750, USA. yc12b@nih.gov

    The regulatory subunit of cAMP-dependent protein kinase (PKA) exists in the isoforms RI and RII, which distinguish PKA isozymes type I (PKA-I) and type II (PKA-II). Evidence obtained from different experimental approaches-such as site-selective cAMP analogs, antisense oligonucleotides, transcription factor decoys, cDNA microarrays, and gene transfer-has shown that PKA-I and -II are expressed in a balance of cell growth and differentiation. Loss of this balance may underlie cancer genesis and progression. DNA microarrays demonstrate that antisense suppression of the RIalpha, which upregulates RIIbeta, downregulates a wide range of genes involved in cell proliferation and transformation while upregulating cell differentiation and reverse transformation genes in PC3M prostate tumors that undergo regression. Conversely, the vector-mediated overexpression of RIIbeta, as opposed to those of RIalpha and Calpha, exhibits induction of differentiation genes along with suppression of cell proliferation and transformation genes leading to reversion of tumor phenotype. Thus, switching of PKA isozyme can cause tumor cells to undergo phenotypic reversion of the malignancy.

    Annals of the New York Academy of Sciences 2005;1058;76-86

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

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

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

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

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

    Nature 2005;437;7062;1173-8

  • A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A.

    Zhang L, Duan CJ, Binkley C, Li G, Uhler MD, Logsdon CD and Simeone DM

    Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    Transforming growth factor beta (TGFbeta) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFbeta signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFbeta activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFbeta activation of CREB, induction of p21(Cip1), and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFbeta and PKA signaling pathways.

    Funded by: NIDDK NIH HHS: 1R03DK60486-01, 5P30 DK34933, DK02137-1, DK41225, P30 DK034933, R01 DK041225

    Molecular and cellular biology 2004;24;5;2169-80

  • HIV-1 gp120 induces anergy in naive T lymphocytes through CD4-independent protein kinase-A-mediated signaling.

    Masci AM, Galgani M, Cassano S, De Simone S, Gallo A, De Rosa V, Zappacosta S and Racioppi L

    Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, 5 via S. Pansini, I-80131 Naples, Italy.

    The ability of the envelope glycoprotein gp120 [human immunodeficiency virus (HIV) env] to induce intracellular signals is thought to contribute to HIV-1 pathogenesis. In the present study, we found that the exposure of CD4+ CD45RA+ naive T cells to HIVenv results in a long-lasting hyporesponsiveness to antigen stimulation. This phenomenon is not dependent on CD4-mediated signals and also can be generated by the exposure of naive T cell to soluble CD4-HIVenv complexes. The analysis of the proximal signaling reveals that HIVenv does not activate Lck as well as the mitogen-activated protein kinase intermediate cascade. Conversely, the envelope glycoprotein stimulates the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity and induces the progressive accumulation of the phosphorylated form of the cAMP-responsive element binding. Of note, the ligation of CXCR4 by stromal cell-derived factor-1alpha but not the engagement of CD4 by monoclonal antibody stimulates the PKA activity and induces a long-lasting hyporesponsivity state in naive CD4+ lymphocytes. The pretreatment of lymphocytes with H89, a cell-permeable PKA inhibitor, prevents the induction of anergy. These findings reveal a novel mechanism by which HIVenv may modulate the processes of clonal expansion, homeostatic proliferation, and terminal differentiation of the naive T lymphocyte subset.

    Journal of leukocyte biology 2003;74;6;1117-24

  • Human myometrial quiescence and activation during gestation and parturition involve dramatic changes in expression and activity of particulate type II (RII alpha) protein kinase A holoenzyme.

    MacDougall MW, Europe-Finner GN and Robson SC

    Department of Obstetrics and Gynaecology, School of Surgical and Reproductive Sciences, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom.

    There are substantial data indicating that components of the cAMP-signaling pathway are differentially expressed in the human myometrium during pregnancy. The effects of cAMP in most tissues and cell types are mainly modulated via protein kinase A, a heterotetrameric protein complex consisting of two regulatory (R) and two catalytic (C) subunits. In the studies presented here, we used specific antibodies in Western blotting/immunoprecipitation, RT-PCR, and functional protein kinase A (PKA) phosphorylation assays to determine the PKA holoenzymes that are expressed in the human myometrium throughout pregnancy and labor. We report that as early as the second trimester of pregnancy, there is a significant increase in expression of the regulatory RII alpha protein subunit of PKA in the myometrium. This increase in protein expression is also mirrored at the mRNA level, indicating transcriptional control throughout pregnancy, whereas during parturition both transcript and protein are significantly decreased. This increase in RII alpha protein also resulted in increased particulate PKA activity in the myometrium during gestation, which was subsequently decreased during labor. Two specific A kinase anchoring proteins, AKAP95 and AKAP79, which have high binding affinities for RII alpha subunits, were found to form complexes with myometrial RII alpha species employing immunoprecipitation assays, but their levels of expression remained uniform in all myometrial tissue samples investigated. Our findings indicate that increased particulate type II PKA activity occurs throughout pregnancy, therefore directing the cAMP quiescence signal to specific subcellular loci within myometrial smooth muscle cells including the contractile machinery at the cytoskeleton; this effect is then removed during parturition.

    The Journal of clinical endocrinology and metabolism 2003;88;5;2194-205

  • Bioinformatic design of A-kinase anchoring protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring.

    Alto NM, Soderling SH, Hoshi N, Langeberg LK, Fayos R, Jennings PA and Scott JD

    Howard Hughes Medical Institute, Vollum Institute, Oregon Health and Science University, Portland OR 97239, USA.

    Compartmentalization of the cAMP-dependent protein kinase (PKA) is coordinated through association with A-kinase anchoring proteins (AKAPs). A defining characteristic of most AKAPs is a 14- to 18-aa sequence that binds to the regulatory subunits (RI or RII) of the kinase. Cellular delivery of peptides to these regions disrupts PKA anchoring and has been used to delineate a physiological role for AKAPs in the facilitation of certain cAMP-responsive events. Here, we describe a bioinformatic approach that yields an RII-selective peptide, called AKAP-in silico (AKAP-IS), that binds RII with a K(d) of 0.4 nM and binds RI with a K(d) of 277 nM. AKAP-IS associates with the type II PKA holoenzyme inside cells and displaces the kinase from natural anchoring sites. Electrophysiological recordings indicate that perfusion of AKAP-IS evokes a more rapid and complete attenuation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents than previously described anchoring inhibitor peptides. Thus, computer-based and peptide array screening approaches have generated a reagent that binds PKA with higher affinity than previously described AKAPs.

    Funded by: NIDDK NIH HHS: P01 DK054441; NIGMS NIH HHS: GM48231, R01 GM048231, R37 GM048231; PHS HHS: DM54441

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;8;4445-50

  • Glucagon and regulation of glucose metabolism.

    Jiang G and Zhang BB

    Department of Metabolic Disorders and Molecular Endocrinology, Merck Research Laboratory, Rahway, New Jersey 07065, USA.

    As a counterregulatory hormone for insulin, glucagon plays a critical role in maintaining glucose homeostasis in vivo in both animals and humans. To increase blood glucose, glucagon promotes hepatic glucose output by increasing glycogenolysis and gluconeogenesis and by decreasing glycogenesis and glycolysis in a concerted fashion via multiple mechanisms. Compared with healthy subjects, diabetic patients and animals have abnormal secretion of not only insulin but also glucagon. Hyperglucagonemia and altered insulin-to-glucagon ratios play important roles in initiating and maintaining pathological hyperglycemic states. Not surprisingly, glucagon and glucagon receptor have been pursued extensively in recent years as potential targets for the therapeutic treatment of diabetes.

    American journal of physiology. Endocrinology and metabolism 2003;284;4;E671-8

  • Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation.

    Ficarro S, Chertihin O, Westbrook VA, White F, Jayes F, Kalab P, Marto JA, Shabanowitz J, Herr JC, Hunt DF and Visconti PE

    Department of Chmeistry, University of Virginia, Charlottesville, Virginia 22908, USA.

    Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.

    Funded by: NICHD NIH HHS: HD 38082; NIGMS NIH HHS: GM 37537; PHS HHS: U54 29099

    The Journal of biological chemistry 2003;278;13;11579-89

  • Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2).

    Li H, Adamik R, Pacheco-Rodriguez G, Moss J and Vaughan M

    Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

    Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately 200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1-832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RI alpha. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RI alpha, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27-48 (domain A) interacted with RI alpha and RI beta, 284-301 (domain B) interacted with RII alpha and RII beta, and 517-538 (domain C) interacted with RI alpha, RII alpha, and RII beta. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;4;1627-32

  • Endosome-to-Golgi transport is regulated by protein kinase A type II alpha.

    Birkeli KA, Llorente A, Torgersen ML, Keryer G, Taskén K and Sandvig K

    Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.

    Studies of RII alpha-deficient B lymphoid cells and stable transfectants expressing the type II alpha regulatory subunit (RII alpha) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type II alpha mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RII alpha-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RII alpha in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type II alpha seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RII alpha expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RII alpha were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type II alpha isozyme.

    The Journal of biological chemistry 2003;278;3;1991-7

  • Characterization of an A-kinase anchoring protein in human ciliary axonemes.

    Kultgen PL, Byrd SK, Ostrowski LE and Milgram SL

    Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

    Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase-anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.

    Funded by: NHLBI NIH HHS: HL-34355, HL-60280, HL-63103, P50 HL060280

    Molecular biology of the cell 2002;13;12;4156-66

  • Exogenous protein kinases A and C, but not endogenous prostasome-associated protein kinase, phosphorylate semenogelins I and II from human semen.

    Ek P, Malm J, Lilja H, Carlsson L and Ronquist G

    Department of Medical Biochemistry and Microbiology, University of Uppsala, Sweden. pia.ek@imbim.uu.se

    Semenogelins I and II are the quantitatively dominating proteins in human semen. They comprise the major part of the sperm-entrapping gel formed at ejaculation, which subsequently liquefies due to proteolysis of the gel-forming proteins by prostate-specific antigen (PSA). The mechanism behind gel formation and its physiological significance is not known. We have studied phosphorylation and dephosphorylation of human semenogelins. Both were phosphorylated by protein kinases A and C (PKA and PKC, respectively) at a rate about 5 times less than that of histone. For PKA, incorporated ((32)P)phosphate into semenogelin approached a maximum above 1 mol/mol. Corresponding values for phosphorylation of the semenogelins with PKC were greater than 10. There was no change in the sensitivity of phosphosemenogelins to proteolysis by PSA. Serine (PKA) and serine and threonine (PKC) were the phosphate-accepting amino acid residues, and all incorporated ((32)P)phosphate could be removed from the semenogelins with human acid phosphatase. Nil or very little phosphate could be detected in purified semenogelins isolated from seminal plasma. In vivo, about half the protein kinase activity in seminal plasma was bound to prostasomes. PKA but not PKC purified from prostasomes could phosphorylate specific substrates, but they could phosphorylate either of the semenogelins.

    Journal of andrology 2002;23;6;806-14

  • A-kinase anchoring protein AKAP220 binds to glycogen synthase kinase-3beta (GSK-3beta ) and mediates protein kinase A-dependent inhibition of GSK-3beta.

    Tanji C, Yamamoto H, Yorioka N, Kohno N, Kikuchi K and Kikuchi A

    Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.

    Glycogen synthase kinase-3 (GSK-3) is regulated by various extracellular ligands and phosphorylates many substrates, thereby regulating cellular functions. Using yeast two-hybrid screening, we found that GSK-3beta binds to AKAP220, which is known to act as an A-kinase anchoring protein. GSK-3beta formed a complex with AKAP220 in intact cells at the endogenous level. Cyclic AMP-dependent protein kinase (PKA) and type 1 protein phosphatase (PP1) were also detected in this complex, suggesting that AKAP220, GSK-3beta, PKA, and PP1 form a quaternary complex. It has been reported that PKA phosphorylates GSK-3beta, thereby decreasing its activity. When COS cells were treated with dibutyryl cyclic AMP to activate PKA, the activity of GSK-3beta bound to AKAP220 decreased more markedly than the total GSK-3beta activity. Calyculin A, a protein phosphatase inhibitor, also inhibited the activity of GSK-3beta bound to AKAP220 more strongly than the total GSK-3beta activity. These results suggest that PKA and PP1 regulate the activity of GSK-3beta efficiently by forming a complex with AKAP220.

    The Journal of biological chemistry 2002;277;40;36955-61

  • The adenosine 2b receptor is recruited to the plasma membrane and associates with E3KARP and Ezrin upon agonist stimulation.

    Sitaraman SV, Wang L, Wong M, Bruewer M, Hobert M, Yun CH, Merlin D and Madara JL

    Division of Digestive Diseases, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA. ssitar2@emory.edu

    We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5'-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane.

    Funded by: NIDDK NIH HHS: DK 35932, DK 47662, K01 DK 02831, K08 DK02802

    The Journal of biological chemistry 2002;277;36;33188-95

  • AMAP-1, a novel testis-specific AMY-1-binding protein, is differentially expressed during the course of spermatogenesis.

    Yukitake H, Furusawa M, Taira T, Iguchi-Ariga SM and Ariga H

    Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan.

    AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with AKAP84/149 in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AMAP-1, was obtained. The amap-1 gene was mapped at human chromosome 17q21. AMY-1 was found to bind to and be colocalized with AMAP-1 in human 293T and HeLa cells. AMAP-1 was found to be specifically expressed in the testis and expressed post-meiotically in the testis, as was AMY-1. These results suggest that both AMAP-1 and AMY-1 play roles in spermatogenesis.

    Biochimica et biophysica acta 2002;1577;1;126-32

  • Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics.

    Alto NM, Soderling J and Scott JD

    Howard Hughes Medical Institute, Vollum Institute, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA.

    A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) and other signaling enzymes to distinct subcellular organelles. Using the yeast two-hybrid approach, we demonstrate that Rab32, a member of the Ras superfamily of small molecular weight G-proteins, interacts directly with the type II regulatory subunit of PKA. Cellular and biochemical studies confirm that Rab32 functions as an AKAP inside cells. Anchoring determinants for PKA have been mapped to sites within the conserved alpha5 helix that is common to all Rab family members. Subcellular fractionation and immunofluorescent approaches indicate that Rab32 and a proportion of the cellular PKA pool are associated with mitochondria. Transient transfection of a GTP binding-deficient mutant of Rab32 promotes aberrant accumulation of mitochondria at the microtubule organizing center. Further analysis of this mutant indicates that disruption of the microtubule cytoskeleton results in aberrantly elongated mitochondria. This implicates Rab32 as a participant in synchronization of mitochondrial fission. Thus, Rab32 is a dual function protein that participates in both mitochondrial anchoring of PKA and mitochondrial dynamics.

    Funded by: NIDDK NIH HHS: DK 54441, P01 DK054441

    The Journal of cell biology 2002;158;4;659-68

  • Novel mechanism of regulation of Rac activity and lamellipodia formation by RET tyrosine kinase.

    Fukuda T, Kiuchi K and Takahashi M

    Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Japan.

    Rac activation in neuronal cells plays an important role in lamellipodia formation that is a critical event for neuritogenesis. It is well known that the Rac activity is regulated via activation of phosphatidylinositol 3-kinase (PI3K) by a variety of receptor tyrosine kinases. Here we show that increased serine phosphorylation on RET receptor tyrosine kinase following cAMP elevation promotes lamellipodia formation of neuronal cells induced by glial cell line-derived neurotrophic factor (GDNF). We identified serine 696 in RET as a putative phosphorylation site by protein kinase A and found that mutation of this serine almost completely inhibited lamellipodia formation by GDNF without affecting activation of the PI3K/AKT signaling pathway. Mutation of tyrosine 1062 in RET, whose phosphorylation is crucial for activation of PI3K, also inhibited lamellipodia formation by GDNF. Inhibition of lamellipodia formation by mutation of either serine 696 or tyrosine 1062 was associated with decrease of the Rac1-guanine nucleotide exchange factor (GEF) activity, suggesting that this activity is regulated by two different signaling pathways via serine 696 and tyrosine 1062 in RET. Moreover, in the presence of serine 696 mutation, lamellipodia formation was rescued by replacing tyrosine 687 with phenylalanine. These findings propose a novel mechanism that receptor tyrosine kinase modulates actin dynamics in neuronal cells via its cAMP-dependent phosphorylation.

    The Journal of biological chemistry 2002;277;21;19114-21

  • Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha.

    Morikis D, Roy M, Newlon MG, Scott JD and Jennings PA

    Department of Chemical and Environmental Engineering, University of California at Riverside, 92093-0359, USA. dmorikis@engr.ucr.edu

    The structure of the N-terminal docking and dimerization domain of the type IIalpha regulatory subunit (RIIalpha D/D) of protein kinase A (PKA) forms a noncovalent stand-alone X-type four-helix bundle structural motif, consisting of two helix-loop-helix monomers. RIIalpha D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein-protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein-protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIalpha D/D and revealed the function of the hydrophobic face as the binding site to A-kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIalpha D/D we have predicted mean apparent pKa values, based on Poisson-Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34-X-X-X-Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter-helical hydrogen bond between Tyr35-Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter-monomer hydrogen bond between Asp30-Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27-Asp30-Glu34-Arg38-Arg40-Glu41-Arg43-Arg44 may be necessary to provide flexibility or stability in the region between the C-terminus and the interdomain/autoinhibitory sequence of RIIalpha, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.

    Funded by: NCI NIH HHS: CA09523; NIDDK NIH HHS: DK07233, DK54441; NIGMS NIH HHS: GM07313, GM19879

    European journal of biochemistry 2002;269;8;2040-51

  • Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes.

    Schillace RV, Andrews SF, Liberty GA, Davey MP and Carr DW

    Veterans Affairs Medical Center and Department of Medicine, Oregon Health and Sciences University, Portland, OR 97201, USA.

    Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.

    Funded by: NIAID NIH HHS: AI10520, F32 AI010520-01A1, F32 AI010520-02

    Journal of immunology (Baltimore, Md. : 1950) 2002;168;4;1590-9

  • Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel.

    Marx SO, Kurokawa J, Reiken S, Motoike H, D'Armiento J, Marks AR and Kass RS

    Department of Pharmacology, Center for Molecular Cardiology, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.

    Sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD) is mediated by beta adrenergic receptor (betaAR) activation, which increases the slow outward potassium ion current (IKS). Mutations in two human I(KS) channel subunits, hKCNQ1 and hKCNE1, prolong APD and cause inherited cardiac arrhythmias known as LQTS (long QT syndrome). We show that betaAR modulation of I(KS) requires targeting of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) and protein phosphatase 1 (PP1) to hKCNQ1 through the targeting protein yotiao. Yotiao binds to hKCNQ1 by a leucine zipper motif, which is disrupted by an LQTS mutation (hKCNQ1-G589D). Identification of the hKCNQ1 macromolecular complex provides a mechanism for SNS modulation of cardiac APD through IKS.

    Funded by: NHLBI NIH HHS: P01HL67849-01, R01-HL44365-07, R01-HL56180, R01-HL56810-05, R01-HL61503, R01-HL68093; NIAID NIH HHS: R01-AI39794

    Science (New York, N.Y.) 2002;295;5554;496-9

  • Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation.

    Niu J, Vaiskunaite R, Suzuki N, Kozasa T, Carr DW, Dulin N and Voyno-Yasenetskaya TA

    Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.

    Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.

    Funded by: NICHD NIH HHS: HD36408; NIGMS NIH HHS: GM56159, GM59427

    Current biology : CB 2001;11;21;1686-90

  • Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells.

    Xie G and Raufman JP

    Department of Internal Medicine, Division of Gastroenterology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205, USA.

    Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187-stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways.

    American journal of physiology. Gastrointestinal and liver physiology 2001;281;4;G1051-8

  • MTG8 proto-oncoprotein interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase in lymphocytes.

    Fukuyama T, Sueoka E, Sugio Y, Otsuka T, Niho Y, Akagi K and Kozu T

    Saitama Cancer Center Research Institute, Saitama 362-0806, Japan.

    AML1-MTG8 chimeric oncogene is generated in acute myelogenous leukemia with t(8;21), and seems to be responsible for the pathogenesis of the disease. However, the role of MTG8 is ambiguous. Here we found that MTG8 interacted with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RIIalpha). The binding site of MTG8 was NHR3 domain, and that of RIIalpha was the N-terminus for interacting with PKA anchoring proteins (AKAPs). NHR3 contains a putative alpha-amphipathic helix which is characteristic in binding of AKAPs with RII. Indirect immunofluorescence microscopy showed that MTG8 and RIIalpha were overlapped at the centrosome-Golgi area in lymphocytes. These findings suggest that MTG8 may function as an AKAP at the centrosome-Golgi area in lymphocytes.

    Oncogene 2001;20;43;6225-32

  • Binding of PKA-RIIalpha to the Adenovirus E1A12S oncoprotein correlates with its nuclear translocation and an increase in PKA-dependent promoter activity.

    Fax P, Carlson CR, Collas P, Taskén K, Esche H and Brockmann D

    Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Essen, Germany.

    The adenovirus type 12 (Ad12) E1A12S oncoprotein utilizes the cAMP/protein kinase A (PKA) signal transduction pathway to activate expression of the viral E2 gene, the products of which are essential for viral replication. A central unsolved question is, however, whether E1A12S interacts directly with PKA in the process of promoter activation. We show here that E1A12S binds to the regulatory subunits (R) of PKA in vitro and in vivo. Interaction depends on the N-terminus and the conserved region 1 (CR1) of E1A12S. Both domains are also essential for the activation of viral E2 gene expression. Infection of cells with Ad12 leads to the cellular redistribution of RIIalpha from the cytoplasm into the nucleus. Furthermore, RIIalpha is also located in the nucleus of cells transformed by E1 of Ad12 and transient expression of E1A12S leads to the redistribution of RIIalpha into the nucleus in a N-terminus- and CR1-dependent manner. Cotransfection of E1A12S with RIIalpha results in strong activation of the E2 promoter. Based on these results we conclude that E1A12S functions as a viral A-kinase anchoring protein redistributing RIIalpha from the cytoplasm into the nucleus where it is involved in E1A12S-mediated activation of the E2 promoter.

    Virology 2001;285;1;30-41

  • Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA.

    Carr DW, Fujita A, Stentz CL, Liberty GA, Olson GE and Narumiya S

    Veterans Affairs Medical Center and Oregon Health Sciences University, Portland, Oregon 97201, USA. carrd@ohsu.edu

    The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoring proteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.

    Funded by: NICHD NIH HHS: HD20419, HD36408

    The Journal of biological chemistry 2001;276;20;17332-8

  • mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module.

    Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, Houslay MD, Langeberg LK and Scott JD

    Howard Hughes Medical Institute and Vollum Institute, and Department of Pediatrics, Oregon Health Sciences University, Portland, OR 97201-3098, USA.

    Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle-selective A-kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram-inhibited cAMP-specific phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP-associated phosphodiesterase activity. Disruption of PKA- mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels.

    Funded by: NHLBI NIH HHS: K08 HL004229, Z01 HL004229; NIDDK NIH HHS: DK54441, P01 DK054441

    The EMBO journal 2001;20;8;1921-30

  • Intracellular distribution of gravin, a PKA and PKC binding protein, in vascular endothelial cells.

    Grove BD and Bruchey AK

    Department of Anatomy and Cellular Biology, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, N. Dak., USA. bgrove@medicine.nodak.edu

    Gravin, a high-molecular-weight protein expressed widely in tissues and cells, is upregulated in cultured endothelial cells under conditions which suggest that it may play a role in wound repair and vascular development. In the current study, we examined the intracellular distribution of gravin to determine if it is associated with the cytoskeleton or with another intracellular compartment. Immunofluorescence microscopy of human umbilical vein endothelial cells (HUVEC) revealed that gravin had a punctate staining distribution that extended to the cell margin and did not appear to colocalize with stress fibers, microtubules, and intermediate filaments. Moreover, disruption of the cytoskeletal structures with either cytochalasin D or colchicine did not alter gravin distribution. However, confocal and immunoelectron microscopy clearly revealed that gravin was concentrated at the cell margin in close association with the plasma membrane. Immunoprecipitation of gravin from endothelial cell lysates resulted in coprecipitation of protein kinase activity that could be eluted from the immunoprecipitates with cAMP and that was inhibitable with a PKA-specific inhibitor. An anti-PKA catalytic subunit antibody reacted with a 40-kD band on immunoblots of the cAMP eluate. Immunoblots of the immunoprecipitates further revealed that PKCalpha coprecipitated with gravin from endothelial cell lysates. This study indicates that gravin is associated with either the plasma membrane or the membrane skeleton and may play a role in endothelial wound healing by targeting PKA and PKC to specific membrane-associated sites and regulating PKA/PKC-dependent cellular activities associated with endothelial wound healing.

    Journal of vascular research 2001;38;2;163-75

  • Selectivity and regulation of A-kinase anchoring proteins in the heart. The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase.

    Zakhary DR, Fink MA, Ruehr ML and Bond M

    Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic alpha-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (K(d)) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 microm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIalpha binding to AKAP100/mAKAP and AKAP15/18 by approximately 7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.

    Funded by: NHLBI NIH HHS: F32HL10236, HL56256; NIA NIH HHS: AG16613; ...

    The Journal of biological chemistry 2000;275;52;41389-95

  • Neurobeachin: A protein kinase A-anchoring, beige/Chediak-higashi protein homolog implicated in neuronal membrane traffic.

    Wang X, Herberg FW, Laue MM, Wullner C, Hu B, Petrasch-Parwez E and Kilimann MW

    Institut für Physiologische Chemie and Institut für Anatomie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.

    We describe the identification and initial characterization of neurobeachin, a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site (K(d), 10 nm) for the type II regulatory subunit of protein kinase A (PKA RII). Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes. It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane. In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTPgammaS and prevented by brefeldin A. Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding. These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat. A neurobeachin isoform that does not bind RII, beige-like protein (BGL), is expressed in many tissues. Neurobeachin, BGL, and approximately 10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family. The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome). Neurobeachin's subcellular localization, its coat protein-like membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;23;8551-65

  • Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold.

    Westphal RS, Soderling SH, Alto NM, Langeberg LK and Scott JD

    Howard Hughes Medical Institute, Vollum Institute, Portland, OR, USA.

    WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl.

    Funded by: NIDDK NIH HHS: DK44239, P01 DK044239

    The EMBO journal 2000;19;17;4589-600

  • PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts.

    Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N and Marks AR

    Center for Molecular Cardiology, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.

    The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major source of calcium (Ca2+) required for cardiac muscle excitation-contraction (EC) coupling. The channel is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). We show that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). Using cosedimentation and coimmunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein, mAKAP. In failing human hearts, RyR2 is PKA hyperphosphorylated, resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

    Funded by: NHLBI NIH HHS: R01 HL56180, R01 HL61503; NIAID NIH HHS: R01 AI39794; ...

    Cell 2000;101;4;365-76

  • Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin.

    Sun F, Hug MJ, Bradbury NA and Frizzell RA

    Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.

    Funded by: NIDDK NIH HHS: DK56490

    The Journal of biological chemistry 2000;275;19;14360-6

  • Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding.

    Herberg FW, Maleszka A, Eide T, Vossebein L and Tasken K

    Ruhr-Universität Bochum, Bochum, 44801, Germany. W.Herberg@ruhr-uni-bochum.de

    Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.

    Journal of molecular biology 2000;298;2;329-39

  • The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis.

    Collas P, Le Guellec K and Taskén K

    Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, Blindern, 0317 Oslo, Norway. philippe.collas@basalmed.uio.no

    Protein kinase A (PKA) and the nuclear A-kinase-anchoring protein AKAP95 have previously been shown to localize in separate compartments in interphase but associate at mitosis. We demonstrate here a role for the mitotic AKAP95-PKA complex. In HeLa cells, AKAP95 is associated with the nuclear matrix in interphase and redistributes mostly into a chromatin fraction at mitosis. In a cytosolic extract derived from mitotic cells, AKAP95 recruits the RIIalpha regulatory subunit of PKA onto chromatin. Intranuclear immunoblocking of AKAP95 inhibits chromosome condensation at mitosis and in mitotic extract in a PKA-independent manner. Immunodepletion of AKAP95 from the extract or immunoblocking of AKAP95 at metaphase induces premature chromatin decondensation. Condensation is restored in vitro by a recombinant AKAP95 fragment comprising the 306-carboxy-terminal amino acids of the protein. Maintenance of condensed chromatin requires PKA binding to chromatin-associated AKAP95 and cAMP signaling through PKA. Chromatin-associated AKAP95 interacts with Eg7, the human homologue of Xenopus pEg7, a component of the 13S condensin complex. Moreover, immunoblocking nuclear AKAP95 inhibits the recruitment of Eg7 to chromatin in vitro. We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.

    The Journal of cell biology 1999;147;6;1167-80

  • mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes.

    Kapiloff MS, Schillace RV, Westphal AM and Scott JD

    Howard Hughes Medical Institute, L-474, Vollum Institute, Portland OR 97201-3098, USA.

    The compartmentalization of second messenger-activated protein kinases contributes to the fidelity of hormone-mediated signal transduction events. For example, the cAMP-dependent protein kinase is tethered at specific intracellular locations through association with A-kinase anchoring proteins (AKAPs). We now report the cloning of mAKAP, an anchoring protein found predominantly in heart, skeletal muscle and brain, and whose expression is induced in neonatal ventriculocytes by treatment with hypertrophic stimuli. mAKAP is targeted to the nuclear membrane of differentiated myocytes. Analysis of mAKAP-green fluorescent protein (GFP) fusion constructs revealed that nuclear membrane targeting is conferred by two regions of the protein, between residues 772-915 and 915-1065, which contain spectrin-like repeat sequences. Heterologous expression of the mAKAP targeting sequences displaced the endogenous anchoring protein from the nuclear membrane, demonstrating that mAKAP targeting is saturable. Collectively, these data suggest that a domain containing spectrin-like repeats mediates targeting of the anchoring protein mAKAP and the cAMP-dependent protein kinase holoenzyme to the nuclear membrane in response to differentiation signals.

    Funded by: NCI NIH HHS: K08 CA69137; NIDDK NIH HHS: DK44239

    Journal of cell science 1999;112 ( Pt 16);2725-36

  • Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus.

    Takahashi M, Shibata H, Shimakawa M, Miyamoto M, Mukai H and Ono Y

    Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan.

    A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.

    The Journal of biological chemistry 1999;274;24;17267-74

  • Diminished levels of protein kinase A RI alpha and RI beta transcripts and proteins in systemic lupus erythematosus T lymphocytes.

    Laxminarayana D, Khan IU, Mishra N, Olorenshaw I, Taskén K and Kammer GM

    Section on Rheumatology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. dlaxmina@wfubmc.edu

    Deficient type I protein kinase A phosphotransferase activity occurs in the T cells of 80% of subjects with systemic lupus erythematosus (SLE). To investigate the mechanism of this deficient isozyme activity, we hypothesized that reduced amounts of type I regulatory (RI) isoform transcripts, RIalpha and RIbeta, may be associated with a diminution of RIalpha and/or RIbeta protein. Sixteen SLE subjects with a mean (+/-1 SD) SLE disease activity index of 12.4 +/- 7.2 were studied. Controls included 16 normal subjects, six subjects with primary Sjögren's syndrome (SS), and three subjects with SS/SLE overlap. RT-PCR revealed that normal, SS, SS/SLE, and SLE T cells expressed mRNAs for all seven R and catalytic (C) subunit isoforms. Quantification of mRNAs by competitive PCR revealed that the ratio of RIalpha mRNA to RIbeta mRNA in normal T cells was 3.4:1. In SLE T cells there were 20 and 49% decreases in RIalpha and RIbeta mRNAs (RIbeta; p = 0.008), respectively, resulting in an RIalpha:RIbeta mRNA of 5.3:1. SS/SLE T cells showed a 72.5% decrease in RIbeta mRNA compared with normal controls (p = 0.01). Immunoblotting of normal T cell RIalpha and RIbeta proteins revealed a ratio of RIalpha:RIbeta of 3.2:1. In SLE T cells, there was a 30% decrease in RIalpha protein (p = 0.002) and a 65% decrease in RIbeta protein (p < 0.001), shifting the ratio of RIalpha:RIbeta protein to 6.5:1. T cells from 25% of SLE subjects lacked any detectable RIbeta protein. Analysis of several lupus T cell lines demonstrated a persistent deficiency of both proteins, excluding a potential effect of disease activity. In conclusion, reduced expression of RIalpha and RIbeta transcripts is associated with a decrement in RIalpha and RIbeta proteins and may contribute to deficient type I protein kinase A isozyme activity in SLE T cells.

    Funded by: NCI NIH HHS: 5P30CA12197-21,21S1; NCRR NIH HHS: MO1RR07122; NIAMS NIH HHS: R01AR39501

    Journal of immunology (Baltimore, Md. : 1950) 1999;162;9;5639-48

  • Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein.

    Vijayaraghavan S, Liberty GA, Mohan J, Winfrey VP, Olson GE and Carr DW

    Kent State University, Ohio 44242, USA.

    Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.

    Funded by: NICHD NIH HHS: HD-36408

    Molecular endocrinology (Baltimore, Md.) 1999;13;5;705-17

  • Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220.

    Schillace RV and Scott JD

    Howard Hughes Medical Institute, L-474 Vollum Institute, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97201-3098, USA.

    The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.

    Funded by: NIDDK NIH HHS: DK48239

    Current biology : CB 1999;9;6;321-4

  • The molecular basis for protein kinase A anchoring revealed by solution NMR.

    Newlon MG, Roy M, Morikis D, Hausken ZE, Coghlan V, Scott JD and Jennings PA

    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0359, USA.

    Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. Formation of these complexes is mediated by specialized protein motifs that participate in protein-protein interactions. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is localized through interaction of the regulatory (R) subunit dimer with A-kinase-anchoring proteins (AKAPs). We now report the solution structure of the type II PKA R-subunit fragment RIIalpha(1-44), which encompasses both the AKAP-binding and dimerization interfaces. This structure incorporates an X-type four-helix bundle dimerization motif with an extended hydrophobic face that is necessary for high-affinity AKAP binding. NMR data on the complex between RIIalpha(1-44) and an AKAP fragment reveals extensive contacts between the two proteins. Interestingly, this same dimerization motif is present in other signaling molecules, the S100 family. Therefore, the X-type four-helix bundle may represent a conserved fold for protein-protein interactions in signal transduction.

    Nature structural biology 1999;6;3;222-7

  • PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I.

    Zimmermann B, Chiorini JA, Ma Y, Kotin RM and Herberg FW

    Institut für Physiologische Chemie I, MA 2/40, Abteilung für Biochemie Supramolekularer Systeme, Medizinische Fakultät der Ruhr-Universität Bochum, D-44801 Bochum, Germany.

    The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.

    The Journal of biological chemistry 1999;274;9;5370-8

  • Identification of tethering domains for protein kinase A type Ialpha regulatory subunits on sperm fibrous sheath protein FSC1.

    Miki K and Eddy EM

    Gamete Biology Group, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

    The fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system and in vitro binding assays with glutathione S-transferase-FSC1 fusion proteins identified two RIalpha tethering domains on FSC1. A domain located at residues 219-232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIalpha tethering site (termed domain B) at residues 335-344 shows specific binding of RIalpha and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIalpha binding. This is apparently the first report of an RIalpha-specific protein kinase A anchoring protein tethering domain.

    The Journal of biological chemistry 1998;273;51;34384-90

  • Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways.

    Zidovetzki R, Wang JL, Chen P, Jeyaseelan R and Hofman F

    Department of Biology and Neuroscience, University of California, Riverside 92521, USA.

    The intracellular signal transduction pathways utilized by the HIV-1-derived protein, Tat, in the activation of human central nervous system-derived endothelial cells (CNS-ECs) were examined using specific enzymatic assays. Tat induced an increase in interleukin 6 (IL-6) mRNA within 1 hr of treatment. This biological effect of Tat involved activation of both protein kinase C (PK-C) and cAMP-dependent protein kinase (PK-A) in CNS-ECs. Tat at 10 ng/ml induced a sharp, transient increase in membrane PK-C activity within 30 sec of incubation, and reached maximum levels at 2 min, declining to control values within 10 min. Tat also induced a sharp increase in intracellular cAMP levels and PK-A activity in these cells, with the PK-A activity reaching a maximum at 10 min and slowly declining to control values in 4 hr of incubation. Activation of PK-A was dependent on a Tat-induced increase in membrane PK-C activity as demonstrated by calphostin C (a PK-C inhibitor) abolishing this effect. Incubation of cells with the cyclooxygenase inhibitor indomethacin did not affect Tat-induced activation of PK-A, indicating that prostacyclins are not involved in this process. Tat-induced increase in IL-6 mRNA was abolished in the presence on PK-A inhibitor H-89, demonstrating that activation of PK-A is necessary and sufficient for the increase in IL-6 production by these cells. Both the Tat-induced increase in intracellular cAMP and IL-6 mRNA levels in CNS-ECs may play a role in altering the blood-brain barrier and thereby inducing pathology often observed in AIDS dementia.

    Funded by: NINDS NIH HHS: NS33805

    AIDS research and human retroviruses 1998;14;10;825-33

  • Mapping of the gene encoding the regulatory subunit RII alpha of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome region 3p21.3-p21.2.

    Taskén K, Naylor SL, Solberg R and Jahnsen T

    Institute of Medical Biochemistry, University of Oslo, Norway. kjetil.tasken@basalmed.uio.no

    We have determined the chromosomal localization of the gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome 3 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. Furthermore, PCR analysis of a chromosome 3 mapping panel revealed the presence of a human RII alpha-specific amplification product only in cell lines containing the region 3p21.3-p21.2. The localization of PRKAR2A was confirmed by PCR mapping using the Stanford G3 Radiation Hybrid Panel as template. The results from this analysis demonstrated that PRKAR2A is most closely linked to D3S3334 (lod score 12.5) and flanked by D3S1322E and D3S1581.

    Funded by: NCI NIH HHS: CA54174, CA56626; PHS HHS: H600470

    Genomics 1998;50;3;378-81

  • HIV-1 tat molecular diversity and induction of TNF-alpha: implications for HIV-induced neurological disease.

    Mayne M, Bratanich AC, Chen P, Rana F, Nath A and Power C

    Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.

    Activation and infection by HIV-1 of glial cells and infiltrating macrophages are cardinal features of AIDS-related neurological disease. Tumor necrosis factor-alpha (TNF-alpha) is released by these cell types, and increased TNF-alpha mRNA and protein levels are associated with the development and severity of HIV-induced neurological disease. HIV-1 proteins have been implicated in HIV neuropathogenesis including Tat which has been shown to be a potent inducer of TNF-alpha. We review our data showing the induction of TNF-alpha by Tat in primary human fetal astrocytes, human peripheral blood mononuclear cells, macrophages, and astrocytic and macrophage cell lines. TNF-alpha induction was NF-kappaB dependent and was eliminated by inhibiting protein kinase A, phospholipase C and protein tyrosine kinase activity. In addition, we examined the molecular diversity of the tat genome in the brains of HIV-infected patients from different HIV-1 clades. Comparison of matched brain- and spleen-derived tat sequences indicated that homology among brain-derived clones was greater than that between the brain- and spleen-derived clones. The brain-derived tat sequences were markedly heterogeneous in regions which influence viral replication and intracellular transport. Future studies using Tat, encoded by different sequences, will be necessary to determine the functional significance of tat molecular diversity. Nonetheless, these studies suggest that Tat is an important inducer of TNF-alpha production and thus may play a key role in the pathogenesis of HIV-related neurological disease.

    Neuroimmunomodulation 1998;5;3-4;184-92

  • Effects of [D-Ala1] peptide T-NH2 and HIV envelope glycoprotein gp120 on cyclic AMP dependent protein kinases in normal and psoriatic human fibroblasts.

    Liapi C, Takahashi N, Raynaud F, Evain-Brion D and Anderson WB

    Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.

    In addition to acquired immunodeficiency syndrome (AIDS), persons infected with human immunodeficiency virus often develop cutaneous manifestations, including severe psoriasis. In previous studies, we have established that psoriatic fibroblasts and erythrocytes obtained from psoriatic patients exhibit decreased levels of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) activity and of 8-azido-[32P]cAMP binding to the RI and RII regulatory subunits of PKA. Because treatment of patients with peptide T (an octapeptide sequence found in the human immunodeficiency virus envelope glycoprotein gp120) has been observed to result in an improvement in the psoriatic condition, studies were initiated to determine if peptide T and gp120 protein treatment of normal and psoriatic human fibroblasts resulted in any changes in PKA. Exposure of psoriatic fibroblasts to peptide T resulted in a time (4 h to 6 d) and dose [10(-14)-10(-8) M] dependent increase in the levels of 8-azido-[32P]cAMP binding to the RI and RII regulatory subunits of PKA, along with a corresponding increase in PKA activity. Peptide T exhibited a biphasic dose dependent response, with maximal effects on PKA noted at 10(-12)M peptide T. Treatment of normal human fibroblasts with peptide T did not result in any change in PKA levels. Conversely, treatment of normal human fibroblasts for 18 h with gp120 protein [10(-13) M] resulted in a significant decrease in the levels of 8-azido-[32P]cAMP binding to RI and RII and in PKA activity. The presence of peptide T blocked this effect of the gp120 protein. These results indicate that peptide T and gp120 protein may inversely alter the intracellular levels of 8-azido-[32P]cAMP binding to RI and RII, and of PKA activity in susceptible cells. These observed changes in the cyclic AMP-PKA signaling pathway, a biochemical marker for psoriasis, may offer some mechanistic insight into the noted beneficial effects of peptide T treatment, including an improvement in psoriatic lesions.

    The Journal of investigative dermatology 1998;110;4;332-7

  • A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor.

    Shetzline MA, Premont RT, Walker JK, Vigna SR and Caron MG

    Howard Hughes Medical Institute, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The secretin receptor is a member of a structurally distinct class of G protein-coupled receptors designated as Class II. The molecular mechanisms of secretin receptor signal termination are unknown. Using transiently transfected HEK 293 cells expressing the secretin receptor, we investigated its mechanisms of desensitization. Binding of [125I]-secretin to plasma membranes of receptor-expressing cells was specific, with a Kd of 2 nM. Secretin evoked an increase in cellular cAMP with an EC50 of 0.4 nM. The response was maximal by 20 min and desensitized rapidly and completely. Immunoprecipitation of a functional, N-terminal epitope-tagged secretin receptor was used to demonstrate agonist-dependent receptor phosphorylation, with an EC50 of 14 nM. Pretreatment with protein kinase A or C inhibitors failed to alter secretin-stimulated cAMP accumulation. G protein-coupled receptor kinases (GRKs) are known to be involved in the desensitization of Class I G protein-coupled receptors; therefore, the effect of cotransfection of GRKs on secretin-stimulated cAMP signaling and phosphorylation was evaluated. GRKs 2 and 5 were the most potent at augmenting desensitization, causing a 40% reduction in the maximal cAMP response to secretin. GRK 5 also caused a shift in the EC50 to the right (p < 0.05). GRK 4 and GRK 6 did not alter dose-dependent signaling, and GRK 3 was intermediate in effect. Receptor phosphorylation correlated with desensitization for each GRK studied, whereas second messenger-dependent kinase phosphorylation appeared to be less important in secretin receptor signal termination. We demonstrate agonist-dependent secretin receptor phosphorylation coincident with profound receptor desensitization of the signaling function in HEK 293 cells, suggesting a role for receptor phosphorylation in this paradigm. Although GRK activity appears important in secretin receptor desensitization in HEK 293 cells, protein kinases A and C appear to play only a minor role. These results demonstrate that the GRK-arrestin system regulates Class II G protein-coupled receptors.

    Funded by: NIDDK NIH HHS: 5T32DK07568; NINDS NIH HHS: NS19576

    The Journal of biological chemistry 1998;273;12;6756-62

  • Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein.

    Dong F, Feldmesser M, Casadevall A and Rubin CS

    Department of Molecular Pharmacology, Atran Laboratories, Bronx, New York 10461, USA.

    We have cloned cDNA that encodes six novel A kinase anchor proteins (collectively named AKAP-KL). AKAP-KL diversity is generated by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL polypeptides are evident in lung, kidney, and cerebellum, but are absent from many tissues. Different isoforms predominate in different tissues. Thus, AKAP-KL expression is differentially regulated in vivo. All AKAP-KL isoforms contain a 20-residue domain that avidly binds (Kd approximately 10 nM) regulatory subunits (RII) of protein kinase AII and is highly homologous with the RII tethering site in neuronal AKAP75. The distribution of AKAP-KL is strikingly asymmetric (polarized) in situ. Anchor protein accumulates near the inner, apical surface of highly polarized epithelium in tubules of nephrons. Both RII and AKAP-KL are enriched at an intracellular site that lies just below the plasma membrane of alveolar epithelial cells in lung. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells. We also demonstrate that the tethering domain of AKAP-KL avidly ligates RII subunits in intact cells. AKAP-KL may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors to capture, amplify, and precisely focus diffuse, trans-cellular signals carried by cAMP.

    Funded by: NIGMS NIH HHS: GM 22792

    The Journal of biological chemistry 1998;273;11;6533-41

  • Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95.

    Eide T, Coghlan V, Orstavik S, Holsve C, Solberg R, Skâlhegg BS, Lamb NJ, Langeberg L, Fernandez A, Scott JD, Jahnsen T and Taskén K

    Institute of Medical Biochemistry, University of Oslo, Blindern, Oslo, N-0317, Norway.

    The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994) J. Biol. Chem. 269, 7658-7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414-692 of human AKAP95 was expressed in Escherichia coli and shown to bind RIIalpha. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIalpha binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIalpha was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIalpha overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIalpha was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIalpha may be cell cycle-dependent.

    Funded by: NIDDK NIH HHS: DK44239, DK52491

    Experimental cell research 1998;238;2;305-16

  • D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain.

    Huang LJ, Durick K, Weiner JA, Chun J and Taylor SS

    Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0654, USA.

    Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

    Funded by: NCI NIH HHS: T32 CA009523, T32 CA09523; NIGMS NIH HHS: 2T32GM07240-21A1, T32 GM007240; NIMH NIH HHS: R01 MH051699, R29MH51699

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;21;11184-9

  • Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase.

    Burton KA, Johnson BD, Hausken ZE, Westenbroek RE, Idzerda RL, Scheuer T, Scott JD, Catterall WA and McKnight GS

    Department of Pharmacology, University of Washington School of Medicine, Box 357750, Seattle, WA 98195-7750, USA.

    Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIalpha subunit would reveal those tissues and signaling events that require anchored PKA. RIIalpha knockout mice appear normal and healthy. In adult skeletal muscle, RIalpha protein levels increased to partially compensate for the loss of RIIalpha. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIalpha knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIalpha subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIalpha subunit. The potentiation of the L-type Ca2+ channel in RIIalpha knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIalpha is capable of physiologically relevant anchoring interactions.

    Funded by: NHLBI NIH HHS: P01 HL044948, P01 HL44948; NICHD NIH HHS: 1 F32 HD08034-01-A1, F32 HD008034; NIDDK NIH HHS: P01 DK044239; NIGMS NIH HHS: GM 32875, R01 GM032875

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;20;11067-72

  • The A-kinase anchoring domain of type IIalpha cAMP-dependent protein kinase is highly helical.

    Newlon MG, Roy M, Hausken ZE, Scott JD and Jennings PA

    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0359, USA.

    Subcellular localization of the type II cAMP-dependent protein kinase is controlled by interaction of the regulatory subunit with A-Kinase Anchoring Proteins (AKAPs). This contribution examines the solution structure of a 44-residue region that is sufficient for high affinity binding to AKAPs. The N-terminal dimerization domain of the type IIalpha regulatory subunit of cAMP-dependent protein kinase was expressed to high levels on minimal media and uniformly isotopically enriched with 15N and 13C nuclei. Sequence-specific backbone and side chain resonance assignments have been made for greater than 95% of the amino acids in the free dimerization domain using high resolution multidimensional heteronuclear NMR techniques. Contrary to the results from secondary structure prediction algorithms, our analysis indicates that the domain is highly helical with a single 3-5-residue sequence involved in a beta-strand. The assignments and secondary structure analysis provide the basis for analyzing the structure and dynamics of the dimerization domain both free and complexed with specific anchoring proteins.

    Funded by: NCI NIH HHS: CA09523; NIDDK NIH HHS: DK44239; NIGMS NIH HHS: GM07313

    The Journal of biological chemistry 1997;272;38;23637-44

  • The Tat protein of HIV-1 induces tumor necrosis factor-alpha production. Implications for HIV-1-associated neurological diseases.

    Chen P, Mayne M, Power C and Nath A

    Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3.

    Human immunodeficiency virus (HIV) infection may cause a dementing illness. HIV-mediated dementia is clinically and pathologically correlated with the infiltration of activated macrophages and elevated levels of tumor necrosis factor (TNF)-alpha, both of which occur in an environment of small numbers of infected cells. We examined the possibility that HIV protein Tat, which is released extracellularly from infected cells, may induce the production of TNF-alpha. Tat induced TNF-alpha mRNA and protein production dose-dependently, primarily in macrophages but also in astrocytic cells. The TNF-alpha induction was NF-kappaB-dependent and could be eliminated by inhibiting protein kinase A or protein tyrosine kinase activity. In addition, Tat-induced TNF-alpha release was also linked to phospholipase C activation. However, Tat effects were independent of protein kinase C. These observations suggest that Tat may provide an important link between HIV and macrophage/glial cell activation and suggest new therapeutic approaches for HIV dementia.

    The Journal of biological chemistry 1997;272;36;22385-8

  • The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix.

    Swingler S, Gallay P, Camaur D, Song J, Abo A and Trono D

    Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

    The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.

    Funded by: NIAID NIH HHS: AI34306, R01 AI37510

    Journal of virology 1997;71;6;4372-7

  • Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits.

    Huang LJ, Durick K, Weiner JA, Chun J and Taylor SS

    Department of Chemistry and Biochemistry, School of Medicine, University of California, San Diego, La Jolla, California 92093-0654, USA.

    Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.

    Funded by: NCI NIH HHS: T32 CA09523; NIGMS NIH HHS: 2T32GM07240-21A1; NIMH NIH HHS: R29MH51699

    The Journal of biological chemistry 1997;272;12;8057-64

  • Molecular cloning, upstream sequence and promoter studies of the human gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase.

    Foss KB, Solberg R, Simard J, Myklebust F, Hansson V, Jahnsen T and Taskén K

    Institute of Medical Biochemistry, University of Oslo, Norway.

    The gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase is highly regulated during spermatogenesis and a strong signal from a distinct short mRNA form is observed postmeiotically during spermatid elongation. This report presents the isolation and characterization of the 5'-flanking region (1.2 kb) and exon 1 of the human RII alpha gene. S1 nuclease mapping and primer extension experiments revealed the presence of a major transcriptional start site located 208 nucleotides upstream of start for translation. The 5'-flanking region of the RII alpha gene did not contain a TATA box and was highly G/C-rich. A basal promoter directing high levels of chloramphenicol acetyl transferase (CAT) activity was identified in the 5'-flanking sequence. Several potential binding sites for transcription factors were identified in this region, which may be responsible for the germ cell-specific regulation of this gene. We have previously reported that the human testis RII alpha cDNA contains a region (amino acids 45-75) with little or no homology to the corresponding rat skeletal muscle cDNA (Oyen, O., Myklebust, F., Scott, J.D., Cadd, G.G., McKnight, G.S., Hansson, V. and Jahnsen, T. (1990) Biol. Reprod. 43, 46-54). We examined whether this difference could arise due to organ-specific splice mechanisms or represented a species difference. We show that the low homology region of the human RII alpha cDNA resides entirely within exon 1, and does not originate from a tissue-specific alternate splicing of this distinct region.

    Biochimica et biophysica acta 1997;1350;1;98-108

  • Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

    Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, Mangeat PH and Goldenring JR

    Institute of Molecular Medicine and Genetics, Medical College of Georgia and the Augusta Veterans Affairs Medical Center, 30912, USA.

    cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.

    Funded by: NIDDK NIH HHS: DK-009026, DK43405

    The EMBO journal 1997;16;1;35-43

  • Cloning and characterization of a novel A-kinase anchoring protein. AKAP 220, association with testicular peroxisomes.

    Lester LB, Coghlan VM, Nauert B and Scott JD

    Vollum Institute, Portland, Oregon 97201, USA.

    Compartmentalization of the type II cyclic AMP-dependent kinase (PKA) is achieved through association of the regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs). Using an interaction cloning strategy with RIIalpha as a probe, we have isolated cDNAs encoding a novel 1129-amino acid protein that contains both a PKA binding region and a peroxisome targeting motif. Northern analysis detected mRNAs of 9.7 and 7.3 kb in several rat tissues with the highest levels present in the brain and testis. Western analysis and RII overlay experiments showed that the protein is approximately 220 kDa and was, therefore, named AKAP 220. Immunoprecipitation of AKAP 220 from rat testis extracts resulted in co-purification of the type II PKA holoenzyme. The specific activity of PKA increased 458-fold from 7.2 pmol/min/mg in the cell lysate to 3.3 nmol/min/mg in the immunoprecipitate. Immunohistochemical analysis of rat testicular TM4 cells showed that AKAP 220 and a proportion of RII were co-localized in microbodies that appear to be a subset of peroxisomes. Collectively, these results suggest that AKAP 220 may play a role in targeting type II PKA for cAMP-responsive peroxisomal events.

    Funded by: NIDDK NIH HHS: DK 02353, DK 09059, DK 44239

    The Journal of biological chemistry 1996;271;16;9460-5

  • Differential localization of two isoforms of the regulatory subunit RII alpha of cAMP-dependent protein kinase in human sperm: biochemical and cytochemical study.

    Pariset C and Weinman S

    Département de Biochimie, UFR Biomédicale des Saints-Pères, Université René Descartes, Paris, France.

    In the present study, immunogold labeling of ultrathin sections of ejaculated sperm was used to obtain insight into the ultrastructural localization and presumable function of type II cAMP-dependent protein kinase in sperm motion. In the flagellum, a human-specific isoform of the RII alpha subunit was located on the axonemal microtubule wall, whereas a different isoform of broader specificity was present in the cytoplasm at the periphery of the coarse fibers and fibrous sheath. This isoform was also found in the mitochondria. The human-specific RII alpha subunit is likely linked to microtubules by a unique binding protein of M(r) 72 kD. These findings are in agreement with the concept of a concerted mechanism involving phosphorylation of both the axonemal microtubules and the fibrous structures for the regulation of mammalian sperm motion.

    Molecular reproduction and development 1994;39;4;415-22

  • Type II regulatory subunit (RII) of the cAMP-dependent protein kinase interaction with A-kinase anchor proteins requires isoleucines 3 and 5.

    Hausken ZE, Coghlan VM, Hastings CA, Reimann EM and Scott JD

    Vollum Institute, Portland, Oregon 97201-3098.

    Compartmentalization of the type II cAMP-dependent protein kinase is maintained by association of the regulatory subunit (RII) with A-Kinase Anchor Proteins (AKAPs). In previous studies (Scott, J. D., Stofko, R. E., McDonald, J. R., Comer, J. D., Vitalis, E. A., and Mangili J. (1990) J. Biol. Chem. 265, 21561-21566) we have shown that dimerization of RII alpha was required for interaction with the cytoskeletal component microtubule-associated protein 2. In this report we show that the localization and dimerization domains of RII alpha are contained within the first thirty residues of each RII protomer. RII des-5 (an amino-terminal deletion mutant lacking residues 1-5) was unable to bind AKAPs but retained the ability to dimerize. RII alpha I3A,I5A (a mutant where isoleucines 3 and 5 were replaced with alanine) was unable to bind a variety of AKAPs. Mutation of both isoleucines decreased AKAP binding without affecting dimerization, cAMP binding, or the overall secondary structure of the protein. Measurement of RII alpha I3A,I5A interaction with the human thyroid AKAP, Ht 31, by two independent methods suggests that mutation of isoleucines 3 and 5 decreases affinity by at least 6-fold. Therefore, we propose that two isoleucine side chains on each RII protomer are principle sites of contact with the conserved amphipathic helix binding domain on AKAPs.

    Funded by: NIDDK NIH HHS: DK 44238

    The Journal of biological chemistry 1994;269;39;24245-51

  • HIV Gag p17 protein impairs proliferation of normal lymphocytes in vitro.

    Hofmann B, Nishanian P, Fan J, Nguyen T and Fahey JL

    Funded by: NIAID NIH HHS: AI 23606, AI 72631

    AIDS (London, England) 1994;8;7;1016-7

  • Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase.

    Coghlan VM, Langeberg LK, Fernandez A, Lamb NJ and Scott JD

    Volum Institute of Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.

    The subcellular location of the type II cAMP-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.

    Funded by: NIDDK NIH HHS: DK44239; NIGMS NIH HHS: GM48231

    The Journal of biological chemistry 1994;269;10;7658-65

  • Type II regulatory subunits of cAMP-dependent protein kinase and their binding proteins in the nervous system of Aplysia californica.

    Cheley S, Panchal RG, Carr DW, Scott JD and Bayley H

    Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.

    Two type II regulatory (R) subunits of cAMP-dependent protein kinase (PKA) of 50 and 47 kDa have been identified in Aplysia neurons by several criteria which include phosphorylation by the catalytic subunit of PKA and nanomolar affinity for a peptide fragment of the human thyroid protein Ht 31, properties that in mammals distinguish type II from type I R subunits. The neuronal type II R subunits are differentially localized within cells. For example, the 50-kDa polypeptide is enriched in taxol-stabilized microtubules. In addition, at least seven high molecular mass neuronal RII-binding proteins ranging in mass from 110 to 420 kDa have been demonstrated by a blot overlay technique, which uses 32P-labeled bovine RII alpha as a probe. The RII-binding proteins also exhibit discrete patterns of subcellular localization. For example, the 420 kDa species is enriched in taxol-stabilized microtubules and therefore may serve to anchor the 50-kDa RII subunit. The localization of PKA through the association of RII subunits with the binding proteins may anchor the multifunctional kinase close to key substrates and thereby contribute to the spatial organization required to mediate the orderly phosphorylation events that underly neuronal modulation.

    Funded by: NIGMS NIH HHS: GM 48231; NINDS NIH HHS: NS26760

    The Journal of biological chemistry 1994;269;4;2911-20

  • Differential expression of cyclic AMP-dependent protein kinase isozymes in normal human melanocytes and malignant melanomas.

    Beebe SJ, Salomonsky P, Holroyd C and Becker D

    Department of Pediatrics, Eastern Virginia Medical School, Norfolk 23510.

    Analysis of cyclic AMP-dependent protein kinase catalytic (C) and regulatory (R) subunits in normal human melanocytes and malignant melanomas demonstrated molar excess of regulatory over catalytic subunit in both cell types. Overall, greater catalytic activity, as a measure of C subunit, and cyclic AMP binding activity, as a measure of R subunit, were detected in metastatic compared to primary melanomas compared to normal human melanocytes. The protein kinase-specific R subunits, expressed in both melanocytes and malignant melanomas, were identified as RI-alpha and RII-alpha. Differences were noted for the ratios of RI/RII and RI holoenzyme/free RI subunit among different melanomas.

    Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 1993;4;12;1005-12

  • Human immunodeficiency virus proteins induce the inhibitory cAMP/protein kinase A pathway in normal lymphocytes.

    Hofmann B, Nishanian P, Nguyen T, Insixiengmay P and Fahey JL

    Center for Interdisciplinary Research in Immunology and Disease, Johnson Comprehensive Cancer Center, University of California, Los Angeles 90024-1747.

    Proliferation of normal T lymphocytes is impaired by human immunodeficiency virus (HIV) proteins. In this paper, we demonstrate important parts of this mechanism. Initially, HIV-induced impairment of proliferation was shown to be an active process involving induction of protein tyrosine kinases in both CD4 and CD8 T cells. Furthermore, the impairment of cell proliferation was demonstrated to be linked to induction of the inhibitory protein kinase A (PKA) pathway by HIV proteins. This induction of PKA was accompanied by an increase in intracellular cAMP, which is necessary for the activation of PKA. Finally, increases in cAMP/PKA activity were shown to induce biochemical changes that impaired proliferation when cells were stimulated with phytohemagglutinin. This was demonstrated by showing that (i) agents, other than HIV proteins, that increase cAMP/PKA activity (cholera toxoid and 8-bromo-cAMP) also decreased T-lymphocyte proliferation; (ii) exposure of lymphocytes to HIV or cholera toxoid led to decreased membrane activity of the proliferation promoter protein kinase C upon stimulation; and (iii) agents that reduced cAMP generation neutralized the effect of HIV proteins and restored lymphocyte proliferation. These studies show that the HIV-induced augmentation of cAMP/PKA activity may be a key part of the mechanism responsible for all or part of the HIV-induced anergy of T lymphocytes.

    Funded by: NIAID NIH HHS: AI23606, AI72631

    Proceedings of the National Academy of Sciences of the United States of America 1993;90;14;6676-80

  • Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

    Carr DW, Hausken ZE, Fraser ID, Stofko-Hahn RE and Scott JD

    Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.

    The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.

    Funded by: NIDDK NIH HHS: DK 08767, DK 44239; NIGMS NIH HHS: GM 44427

    The Journal of biological chemistry 1992;267;19;13376-82

  • Identification, purification, and characterization of subunits of cAMP-dependent protein kinase in human testis. Reverse mobilities of human RII alpha and RII beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with rat and bovine RIIs.

    Skålhegg BS, Landmark B, Foss KB, Lohmann SM, Hansson V, Lea T and Jahnsen T

    Institute of Pathology, Rikshospitalet, Oslo, Norway.

    We have previously identified and characterized regulatory (R) subunits of cyclic AMP-dependent protein kinase, particularly the RII subunits in rat tissues (Jahnsen, T., Lohmann, S. M., Walter, U., Hedin, L., and Richards, J. S. (1985) J. Biol. Chem. 260, 15980-15987; Jahnsen, T., Hedin, L., Lohmann, S. M., Walter, U., and Richards, J. S. (1986) J. Biol. Chem. 261, 6637-6639; Jahnsen, T., Hedin, L., Kidd, V. J., Beattie, W. G., Lohmann, S. M., Walter, U., Durica, J., Schulz, T. Z., Schiltz, E., Browner, M., Lawrence, C. B., Goldman, D., Ratoosh, S. L., and Richards, J. S. (1986) J. Biol. Chem. 261, 12352-12361). These studies showed that rat RII alpha and RII beta had apparent molecular masses of 54 and 52 kDa, respectively. The aim of the present study was to purify and characterize cAMP-dependent protein kinase R subunits in human testis and to examine which of the subunits (mRNAs and proteins) are present in this tissue. Our results show that human testis contains mRNAs for five out of the seven known subunits of cAMP-dependent protein kinase. We observed strong expression of mRNAs for RI alpha (1.5 and 3.2 kilobases (kb)), RII alpha (2.2, 2.4, and 7.0 kb), and RII beta (3.3 kb). We also demonstrated mRNAs for two of the three catalytic subunits, C alpha (2.7 kb) and C gamma (1.7 kb). Purification of R subunits by DEAE-cellulose and cAMP affinity chromatography revealed three distinct forms with apparent molecular masses of 49, 51, and 53 kDa, respectively. Characterization of these R subunits by their 8-azido-cAMP photoaffinity labeling and immunoreactivity, as well as by a phosphorylation-dependent mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated subunit sizes of RII beta (53 kDa) greater than RII alpha dephosphoform (51 kDa) greater than RI alpha (49 kDa). This conclusion was verified by the analysis of RII subunits produced by in vitro transcription/translation of full-length cDNAs for both human RII alpha and RII beta in wheat germ lysates. The in vitro translated products were the same size as the purified human testis subunits, and only the smallest RII subunit (RII alpha) revealed a distinct mobility shift on SDS-PAGE after phosphorylation/dephosphorylation. This study supports the conclusion that the mobilities of human RII subunits (RII alpha, RII beta) on SDS-PAGE are reversed in contrast with those of other species such as rat and bovine.(ABSTRACT TRUNCATED AT 400 WORDS)

    The Journal of biological chemistry 1992;267;8;5374-9

  • Type II regulatory subunit dimerization determines the subcellular localization of the cAMP-dependent protein kinase.

    Scott JD, Stofko RE, McDonald JR, Comer JD, Vitalis EA and Mangili JA

    Vollum Institute for Advanced Biomedical Research, Portland, Oregon 97201.

    The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of dimeric regulatory subunits (RII) to anchoring proteins. Cytoskeletal localization occurs through RII dimer interaction with the PKA substrate molecule microtubule-associated protein 2 (MAP2). RII alpha deletion mutants and RII alpha/endonexin chimeras retained MAP2 binding activity if they contained the first 79 residues of the molecule. Disruption of RII alpha dimerization always prevented MAP2 interaction because 1) RII delta 1-14 (an amino-terminal deletion mutant lacking residues 1-14) was unable to bind MAP2 or form dimers, and 2) a modified RII alpha monomer including residues 1-14 did not bind MAP2. Chimeric proteins containing the first 30 residues of RII alpha fused to endonexin II formed dimers but did not bind MAP2. This suggested other side-chains between residues 30-79 also participate in MAP2 interaction. Peptide studies indicate additional contact with MAP2 may occur through an acidic region (residues 68-82) close to the RII autoinhibitor domain. Therefore, anchored PKA holoenzyme topology may position the catalytic subunit and MAP2 as to allow its preferential phosphorylation upon kinase activation.

    Funded by: NIGMS NIH HHS: GM 44427

    The Journal of biological chemistry 1990;265;35;21561-6

  • HIV inhibits the early steps of lymphocyte activation, including initiation of inositol phospholipid metabolism.

    Hofmann B, Nishanian P, Baldwin RL, Insixiengmay P, Nel A and Fahey JL

    Center for Interdisciplinary Research in Immunology and Disease (CIRID), UCLA School of Medicine.

    Mechanisms accounting for HIV-associated suppression of lymphocyte proliferation were investigated. In previous work we demonstrated that purified and inactivated HIV-suppressed lymphoid cell proliferation. In this report we used an inactivated preparation of HIV obtained from infected CEM cells grown in serum free media and demonstrated that this HIV-associated suppression acted in the early steps of activation to inhibit the incorporation of radiolabeled phosphorus into phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. Initially we showed that both purified CD4 and CD8 T lymphocyte subsets were affected and HIV-associated inhibition did not require the CD4 molecule. Impaired lymphocyte blastogenesis (decreased size and granularity and decreased expression of receptors to IL-2 and transferrin) in response to PHA indicated an effect of inactivated HIV on the early steps of activation. This was confirmed by time studies where 1) a 2 min HIV-pretreatment followed by washing before stimulation was sufficient to inhibit PHA induced proliferation of normal lymphocytes, and 2) addition of HIV to PHA prestimulated lymphocytes failed to inhibit proliferation, e.g., there was no effect on preactivated lymphocytes. HIV was mainly inhibitory of lymphocyte proliferation induced by PHA or mAb to the CD3 receptor. In contrast to the effect on the CD3/TiR, responses via the CD2 receptor were not suppressed, e.g., stimulation with the monoclonal antibodies T11(2) + T11(3). Inasmuch as responses by direct A23187 + PMA stimulation of intracellular pathways were also inhibited, it appears that the HIV-induced defect was not (or not only) membrane receptor mediated. The earliest (min) measurable event after stimulation was the initial increase in intracellular Ca2+ which was unaffected by HIV pretreatment. The next measurable event (min to h) of stimulation is a sustained increase in inositol phospholipid turnover. Pretreatment of mononuclear cells with inactivated HIV resulted in a decreased inositol phospholipid turnover as judged from decreased 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. This led to decreased generation of DAG as reflected in the reduced radiolabeling of its metabolite PA. Reduced availability of DAG presumably interferes with pkC activation and leads to decreased expression of receptors for IL-2 and transferrin and impaired proliferation.

    Funded by: NHLBI NIH HHS: 2 T32 HL07386; NIAID NIH HHS: AI 23606, AI 72631

    Journal of immunology (Baltimore, Md. : 1950) 1990;145;11;3699-705

  • cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes.

    Taylor SS, Buechler JA and Yonemoto W

    Department of Chemistry, University of California, San Diego, La Jolla 92093.

    cAPK has provided many insights into the functioning of the diverse family of eukaryotic protein kinases. The fact that a particular amino acid in the catalytic core is conserved is an indication that the residue plays an important role; however, questions concerning function remain obscure. With the catalytic subunit, the assignment of amino acids that participate in catalysis has begun, and in many instances that function appears to be conserved in the other protein kinases. Although the regulatory subunit and the use of cAMP to release its inhibitor effects is unique to cAPK, the general mechanism of a small autoinhibitory region occupying the peptide binding site and thus preventing access of other substrates may be invoked frequently by other protein kinases. Coupling recombinant approaches with protein chemistry is allowing us to decipher at least some of the molecular events associated with cAMP-binding and holoenzyme activation. Although the next chapter in the history of cAPK will undoubtedly include three-dimensional structures, the chemical information remains as an essential complement for interpreting those structures and eventually understanding the molecular events associated with catalysis and activation.

    Funded by: NIADDK NIH HHS: AM07233; NIGMS NIH HHS: GM19301, GM34921

    Annual review of biochemistry 1990;59;971-1005

  • The RII subunit of cAMP-dependent protein kinase binds to a common amino-terminal domain in microtubule-associated proteins 2A, 2B, and 2C.

    Obar RA, Dingus J, Bayley H and Vallee RB

    Cell Biology and Neurobiology Groups, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.

    Three products of the MAP2 gene are known: MAP2A and MAP2B (Mr approximately 200,000) and MAP2C (Mr 70,000). The structural relationship between these MAPs and the basis for their diversity in size are unknown. Previously, we found that a significant fraction of type II cAMP-dependent protein kinase was associated via its regulatory subunits with MAP2A and MAP2B. We now use an antibody prepared against the microtubule binding domain of MAP2A and MAP2B to identify MAP2C. All three forms of MAP2 bound to cAMP affinity columns and reacted with 32P-labeled RII in a blot overlay assay. By assaying proteolytic fragments of MAP2A and MAP2B as well as segments of MAP2 expressed in E. coli, the binding site for RII was localized to an 83 amino acid stretch at the distal (amino-terminal) end of the MAP2 arm domain. Therefore, the microtubule binding and RII binding domains are located at extreme opposite ends of MAP2A and MAP2B, and both are conserved in the much shorter MAP2C.

    Funded by: NICHD NIH HHS: HD07312; NIGMS NIH HHS: GM26701

    Neuron 1989;3;5;639-45

  • Localization and characterization of the binding site for the regulatory subunit of type II cAMP-dependent protein kinase on MAP2.

    Rubino HM, Dammerman M, Shafit-Zagardo B and Erlichman J

    Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461.

    Microtubule-associated protein 2 (MAP2) binds, and is a substrate for, type II cAMP-dependent protein kinase. The structural domain in MAP2 that binds the regulatory subunit (RII) of protein kinase II was identified by expressing fragments of a human MAP2 cDNA in E. coli using the pATH11 vector. Fusion proteins were resolved by SDS-PAGE and transferred to nitrocellulose. The filters were probed with purified bovine heart or brain RII, anti-RII monoclonal antibodies, and 125I-labeled protein A. Binding of RII was localized to a 31 amino acid sequence near the N-terminus of the MAP2 molecule. Fusion proteins containing this fragment bound both heart and brain RIIs in a concentration-dependent manner, but bound heart RII with a higher apparent affinity than brain RII. The amino acid sequence of this fragment (DRETAEEVSARIVQVVTAEAVAVLKGEQEKE) is totally conserved between human and mouse MAP2, suggesting an important role for the RII binding site of MAP2 in neuronal function.

    Funded by: NIA NIH HHS: AG-06803; NIDDK NIH HHS: DK-27736

    Neuron 1989;3;5;631-8

  • Human testis cDNA for the regulatory subunit RII alpha of cAMP-dependent protein kinase encodes an alternate amino-terminal region.

    Oyen O, Myklebust F, Scott JD, Hansson V and Jahnsen T

    Institute of Pathology, Rikshospitalet, Oslo, Norway.

    Phosphorylations catalyzed by cAMP-dependent protein kinase are essential for sperm motility, and type II cAMP-dependent protein kinase in mature sperm has been shown to be firmly bound to the flagellum via the regulatory subunit, RII. The present study documents high-levelled expression of a human, testis-specific RII alpha mRNA (2.0 kb) analogous to the rat mRNA which is induced in haploid germ cells [(1988) FEBS Lett. 229, 391-394]. We report the molecular cloning of a full-length human cDNA corresponding to this unique testis mRNA, and the presence of an alternative amino-terminal region (amino acids 45-75) of the predicted RII alpha protein (404 amino acids) compared with the previously published mouse and rat sequences. However, this alternate region is also shown to be present in RII alpha mRNA (7.0 kb) of human somatic cells. Our data indicate the divergent amino-terminal sequence to be due to species differences, suggesting an active evolutionary pressure on this particular region, which could be involved in subcellular attachment of RII alpha and thereby localization of kinase activity to certain targets within the cell.

    FEBS letters 1989;246;1-2;57-64

  • Phosphorylation of the type-II regulatory subunit of cyclic-AMP-dependent protein kinase by glycogen synthase kinase 3 and glycogen synthase kinase 5.

    Hemmings BA, Aitken A, Cohen P, Rymond M and Hofmann F

    European journal of biochemistry 1982;127;3;473-81

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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