G2Cdb::Gene report

Gene id
G00001411
Gene symbol
PRKACB (HGNC)
Species
Homo sapiens
Description
protein kinase, cAMP-dependent, catalytic, beta
Orthologue
G00000162 (Mus musculus)

Databases (7)

Gene
ENSG00000142875 (Ensembl human gene)
5567 (Entrez Gene)
182 (G2Cdb plasticity & disease)
PRKACB (GeneCards)
Literature
176892 (OMIM)
Marker Symbol
HGNC:9381 (HGNC)
Protein Sequence
P22694 (UniProt)

Synonyms (1)

  • PKACb

Literature (43)

Pubmed - other

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

    Shelton RC, Hal Manier D and Lewis DA

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

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

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

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

  • Overexpressing PKIB in prostate cancer promotes its aggressiveness by linking between PKA and Akt pathways.

    Chung S, Furihata M, Tamura K, Uemura M, Daigo Y, Nasu Y, Miki T, Shuin T, Fujioka T, Nakamura Y and Nakagawa H

    Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

    Prostate cancer (PC) is the most common malignancy in males. Despite high response rates and clinical benefits, androgen-ablation therapy is ineffective for advanced or relapsed PC because of the emergence of aggressive castration-resistant prostate cancer (CRPC). Through our genome-wide gene expression analysis of PC cells purified from clinical CRPC tissues, we here identified a novel molecular target, PKIB (cAMP-dependent protein kinase inhibitor-beta), which was overexpressed specifically in CRPCs and aggressive PCs. Immunohistochemical analysis confirmed its overexpression in CRPCs and its strong correlation with high Gleason scores of PCs. Knockdown of PKIB by siRNA resulted in drastic growth suppression of PC cells, and, concordantly, exogenous introduction of PKIB into PC cells enhanced their growth and mobility. We found the direct interaction between PKIB and cAMP-dependent protein kinase A catalytic subunit (PKA-C), and showed that knockdown of PKIB in PC cells diminished the nuclear translocation of PKA-C. Knockdown of PKIB also decreased the phosphorylation level of Akt at Ser473 in PC cells, and exogenous PKIB introduction enhanced Akt phosphorylation in PC cells by incorporating with endogenous PKA-C kinase. In vitro kinase assay validated the recombinant PKIB enhanced phosphorylation of Akt at Ser473 by PKA-C kinase. These findings show that PKIB and PKA-C kinase can have critical functions of aggressive phenotype of PCs through Akt phosphorylation and that they should be a promising molecular target for PC treatment.

    Oncogene 2009;28;32;2849-59

  • PKA-mediated stabilization of FoxH1 negatively regulates ERalpha activity.

    Yum J, Jeong HM, Kim S, Seo JW, Han Y, Lee KY and Yeo CY

    Department of Life Science and Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea.

    Estrogen receptor alpha (ERalpha) mediates the mitogenic effects of estrogen. ERalpha signaling regulates the normal growth and differentiation of mammary tissue, but uncontrolled ERalpha activation increases the risk to breast cancer. Estrogen binding induces ligand-dependent ERalpha activation, thereby facilitating ERalpha dimerization, promoter binding and coactivator recruitment. ERalpha can also be activated in a ligand-independent manner by many signaling pathways, including protein kinase A (PKA) signaling. However, in several ERalpha-positive breast cancer cells, PKA inhibits estrogen-dependent cell growth. FoxH1 represses the transcriptional activities of estrogen receptors and androgen receptors (AR). Interestingly, FoxH1 has been found to inhibit the PKA-induced and ligand-induced activation of AR. In the present study, we examined the effects of PKA activation on the ability of FoxH1 to represses ERalpha transcriptional activity. We found that PKA increases the protein stability of FoxH1, and that FoxH1 inhibits PKA-induced and estradiol-induced activation of an estrogen response element (ERE). Furthermore, in MCF7 cells, FoxH1 knockdown increased the PKA-induced and estradiol-induced activation of the ERE. These results suggest that PKA can negatively regulate ERalpha, at least in part, through FoxH1.

    Molecules and cells 2009;28;1;67-71

  • The paradoxical increase in cortisol secretion induced by dexamethasone in primary pigmented nodular adrenocortical disease involves a glucocorticoid receptor-mediated effect of dexamethasone on protein kinase A catalytic subunits.

    Louiset E, Stratakis CA, Perraudin V, Griffin KJ, Libé R, Cabrol S, Fève B, Young J, Groussin L, Bertherat J and Lefebvre H

    Institut National de la Santé et de la Recherche Médicale, Unité 413, EA4310, Laboratory of Differentiation and Neuronal and Neuroendocrine Communication, University of Rouen, Mont Saint Aignan, France.

    Context: Primary pigmented nodular adrenocortical disease (PPNAD) results in most cases from mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene. Patients with PPNAD exhibit a paradoxical increase in cortisol secretion in response to dexamethasone.

    Objective: The aim was to investigate the mechanism of the action of dexamethasone on adrenocortical cells removed from patients with PPNAD and a transgenic model of PPNAD [Tg(tTA/X2AS) mice].

    We performed an in vitro study in an academic research laboratory.

    Patients: Eleven patients with histologically proven PPNAD were included in the study.

    Intervention: Cultured PPNAD cells were incubated with dexamethasone in the presence of various modulators of the cAMP/PKA pathway and the glucocorticoid receptor antagonist RU486.

    Cortisol and corticosterone were measured by radioimmunological assays in cell culture supernatants.

    Results: Dexamethasone stimulated in vitro cortisol secretion from PPNAD tissues in six patients. The stimulatory effect of dexamethasone on cortisol release was not reduced by the adenylyl cyclase inhibitor SQ22536 or potentiated by the phosphodiesterase inhibitor IMBX and the cAMP analog 8Br-cAMP. Conversely, the PKA inhibitor H89 and RU486 inhibited the cortisol response to dexamethasone. Dexamethasone had no effect on cortisol production from normal human adrenocortical cells but stimulated corticosteroidogenesis in the presence of RU486. Similarly, dexamethasone failed to influence corticosterone release by adrenocortical cells removed from Tg(tTA/X2AS) mice but stimulated corticosteroidogenesis in the presence of RU 486.

    Conclusions: These results indicate that, in human PPNAD tissues, dexamethasone paradoxically stimulates cortisol release through a glucocorticoid receptor-mediated effect on PKA catalytic subunits.

    Funded by: NICHD NIH HHS: Z01 HD000642, Z01-HD-000642-04

    The Journal of clinical endocrinology and metabolism 2009;94;7;2406-13

  • Recruitment of coactivator glucocorticoid receptor interacting protein 1 to an estrogen receptor transcription complex is regulated by the 3',5'-cyclic adenosine 5'-monophosphate-dependent protein kinase.

    Fenne IS, Hoang T, Hauglid M, Sagen JV, Lien EA and Mellgren G

    Institute of Medicine, Section for Endocrinology, University of Bergen, Bergen, Norway.

    Steroid receptor coactivators (SRCs), such as glucocorticoid receptor interacting protein 1 (GRIP1) are recruited to the DNA-bound nuclear receptors (NRs) and are also shown to enhance the gene transactivation by other transcription factors. In contrast to the two other members of the SRC family, SRC-1 and SRC-3/amplified in breast cancer 1, SRC-2/GRIP1 is regulated by the cAMP-dependent protein kinase [protein kinase A (PKA)] that stimulates its ubiquitination and degradation. In this report we demonstrate that COS-1 and MCF-7 cells treated with cAMP-elevating agents and 8-para-chlorophenylthio-cAMP for short periods of time showed an increase in GRIP1 coactivator function, whereas prolonged stimulation of the cAMP/PKA pathway led to a decline in GRIP1-mediated activation and protein levels. Furthermore, MCF-7 breast cancer cells were subjected to chromatin immunoprecipitation assays after stimulation of the cAMP/PKA pathway. cAMP/PKA initiated a rapid recruitment of GRIP1 to the endogenous estrogen receptor (ER)-alpha target pS2 gene promoter. In contrast to the estradiol-induced recruitment of GRIP1 to pS2, we observed an additional increase in GRIP1 recruitment on inhibition of the proteasome, suggesting that inhibition of GRIP1 degradation leads to accumulation at the pS2. Real-time PCR experiments confirmed that cAMP/PKA enhanced the expression of pS2. Moreover, confocal imaging of COS-1 cells transfected with yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ERalpha revealed that PKA led to redistribution and colocalization of yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ERalpha in subnuclear foci. In conclusion, these results suggest that activation of the cAMP/PKA pathway stimulates recruitment of GRIP1 to an ER-responsive gene promoter. The initial stimulation of GRIP1 coactivator function is followed by an increased turnover and subsequent degradation of GRIP1 protein.

    Endocrinology 2008;149;9;4336-45

  • Protein kinase C-beta and -delta isoenzymes promote arachidonic acid production and proliferation of MonoMac-6 cells.

    Griger Z, Páyer E, Kovács I, Tóth BI, Kovács L, Sipka S and Bíró T

    Department of Physiology, University of Debrecen, Research Center for Molecular Medicine, Medical and Health Science Center, Nagyerdei krt. 98., 4032, Debrecen, Hungary.

    In this study, we investigated the putative roles of certain protein kinase C (PKC) isoenzymes in the regulation of proliferation and arachidonic acid (AA) release in the human monocytoid MonoMac-6 cell line. Experiments employing specific PKC inhibitors and molecular biological methods (RNA-interference, recombinant overexpression) revealed that the two dominantly expressed isozymes, i.e., the "conventional" cPKCbeta and the "novel" nPKCdelta, promote AA production and cellular proliferation. In addition, using different phospholipase A(2) (PLA(2)) inhibitors, we were able to show that the calcium-independent iPLA(2) as well as diacylglycerol lipase (but not the cytosolic PLA(2)) function as "downstream" targets of cPKCbeta and nPKCdelta. In addition, we have also found that, among the other existing PKC isoforms, cPKCalpha plays a minor inhibitory role, whereas nPKCvarepsilon and aPKCzeta apparently do not regulate these cellular processes. In conclusion, in this paper we provide the first evidence that certain PKC isoforms play pivotal, specific, and (at least partly) antagonistic roles in the regulation of AA production and cellular proliferation of human monocytoid MonoMac-6 cells.

    Journal of molecular medicine (Berlin, Germany) 2007;85;9;1031-42

  • Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing.

    Kvissel AK, Ørstavik S, Eikvar S, Brede G, Jahnsen T, Collas P, Akusjärvi G and Skålhegg BS

    Department of Nutrition, University of Oslo, Oslo, Norway. a.k.kvissel@basalmed.uio.no

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Calpha and Cbeta are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.

    Experimental cell research 2007;313;13;2795-809

  • Androgen dependent regulation of protein kinase A subunits in prostate cancer cells.

    Kvissel AK, Ramberg H, Eide T, Svindland A, Skålhegg BS and Taskén KA

    Department of Nutrition, University of Oslo, N-0317 Oslo, Norway.

    Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cbeta isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cbeta splice variants are differentially regulated during this process. Whereas the Cbeta2 splice variant is down-regulated in growth arrested LNCaP cells, the Cbeta1, Cbeta3 and Cbeta4 variants, as well as the RIIbeta subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cbeta expression could be mimicked by androgen stimulation, implying the Cbeta gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cbeta subunits were also observed in human prostate specimens, and the Cbeta2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cbeta isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.

    Cellular signalling 2007;19;2;401-9

  • Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cbeta2.

    Funderud A, Henanger HH, Hafte TT, Amieux PS, Orstavik S and Skålhegg BS

    Department of Nutrition Research, Institute of Basic Medical Sciences, University of Oslo, PO Box 1046 Blindern, 0317 Oslo, Norway. ane.funderud@medisin.uio.no

    Background: Two main genes encoding the catalytic subunits Calpha and Cbeta of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cbeta genes encode several splice variants, including the splice variant Cbeta2. In mouse Cbeta2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cbeta2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues.

    Results: We identified a novel 47 kDa splice variant encoded by the mouse Cbeta gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cbeta2. Based on the present results and the existence of a human brain-specifically expressed Cbeta splice variant designated Cbeta4 that is identical to the former mouse Cbeta2 splice variant, the mouse splice variant has now been renamed mouse Cbeta4.

    Conclusion: Murine lymphoid tissues express a protein that is a homologue of human and bovine Cbeta2. The murine Cbeta gene encodes the splice variants Cbeta1, Cbeta2, Cbeta3 and Cbeta4, as is the case with the human Cbeta gene.

    BMC biochemistry 2006;7;20

  • The DNA sequence and biological annotation of human chromosome 1.

    Gregory SG, Barlow KF, McLay KE, Kaul R, Swarbreck D, Dunham A, Scott CE, Howe KL, Woodfine K, Spencer CC, Jones MC, Gillson C, Searle S, Zhou Y, Kokocinski F, McDonald L, Evans R, Phillips K, Atkinson A, Cooper R, Jones C, Hall RE, Andrews TD, Lloyd C, Ainscough R, Almeida JP, Ambrose KD, Anderson F, Andrew RW, Ashwell RI, Aubin K, Babbage AK, Bagguley CL, Bailey J, Beasley H, Bethel G, Bird CP, Bray-Allen S, Brown JY, Brown AJ, Buckley D, Burton J, Bye J, Carder C, Chapman JC, Clark SY, Clarke G, Clee C, Cobley V, Collier RE, Corby N, Coville GJ, Davies J, Deadman R, Dunn M, Earthrowl M, Ellington AG, Errington H, Frankish A, Frankland J, French L, Garner P, Garnett J, Gay L, Ghori MR, Gibson R, Gilby LM, Gillett W, Glithero RJ, Grafham DV, Griffiths C, Griffiths-Jones S, Grocock R, Hammond S, Harrison ES, Hart E, Haugen E, Heath PD, Holmes S, Holt K, Howden PJ, Hunt AR, Hunt SE, Hunter G, Isherwood J, James R, Johnson C, Johnson D, Joy A, Kay M, Kershaw JK, Kibukawa M, Kimberley AM, King A, Knights AJ, Lad H, Laird G, Lawlor S, Leongamornlert DA, Lloyd DM, Loveland J, Lovell J, Lush MJ, Lyne R, Martin S, Mashreghi-Mohammadi M, Matthews L, Matthews NS, McLaren S, Milne S, Mistry S, Moore MJ, Nickerson T, O'Dell CN, Oliver K, Palmeiri A, Palmer SA, Parker A, Patel D, Pearce AV, Peck AI, Pelan S, Phelps K, Phillimore BJ, Plumb R, Rajan J, Raymond C, Rouse G, Saenphimmachak C, Sehra HK, Sheridan E, Shownkeen R, Sims S, Skuce CD, Smith M, Steward C, Subramanian S, Sycamore N, Tracey A, Tromans A, Van Helmond Z, Wall M, Wallis JM, White S, Whitehead SL, Wilkinson JE, Willey DL, Williams H, Wilming L, Wray PW, Wu Z, Coulson A, Vaudin M, Sulston JE, Durbin R, Hubbard T, Wooster R, Dunham I, Carter NP, McVean G, Ross MT, Harrow J, Olson MV, Beck S, Rogers J, Bentley DR, Banerjee R, Bryant SP, Burford DC, Burrill WD, Clegg SM, Dhami P, Dovey O, Faulkner LM, Gribble SM, Langford CF, Pandian RD, Porter KM and Prigmore E

    The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. sgregory@chg.duhs.duke.edu

    The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

    Funded by: Medical Research Council: G0000107; Wellcome Trust

    Nature 2006;441;7091;315-21

  • The LIFEdb database in 2006.

    Mehrle A, Rosenfelder H, Schupp I, del Val C, Arlt D, Hahne F, Bechtel S, Simpson J, Hofmann O, Hide W, Glatting KH, Huber W, Pepperkok R, Poustka A and Wiemann S

    Division Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. a.mehrle@dkfz.de

    LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.

    Nucleic acids research 2006;34;Database issue;D415-8

  • 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

  • Identification of protein kinase A catalytic subunit beta as a novel binding partner of p73 and regulation of p73 function.

    Hanamoto T, Ozaki T, Furuya K, Hosoda M, Hayashi S, Nakanishi M, Yamamoto H, Kikuchi H, Todo S and Nakagawara A

    Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan.

    Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit beta (PKA-Cbeta) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73alpha associated with PKA-Cbeta in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73alpha. In contrast, p53 failed to bind to PKA-Cbeta. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73alpha-(1-130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cbeta resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73alpha, whereas a kinase-deficient mutant of PKA-Cbeta had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73alpha. Of particular interest, PKA-Cbeta facilitated the intramolecular interaction of p73alpha, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cbeta-mediated inhibitory mechanism of p73 function.

    The Journal of biological chemistry 2005;280;17;16665-75

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

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

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

    Funded by: PHS HHS: N01-C0-12400

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

  • From ORFeome to biology: a functional genomics pipeline.

    Wiemann S, Arlt D, Huber W, Wellenreuther R, Schleeger S, Mehrle A, Bechtel S, Sauermann M, Korf U, Pepperkok R, Sültmann H and Poustka A

    Molecular Genome Analysis, German Cancer Research Center, 69120 Heidelberg, Germany. s.wiemann@dkfz.de

    As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

    Genome research 2004;14;10B;2136-44

  • Protein kinase A in postmortem brain of depressed suicide victims: altered expression of specific regulatory and catalytic subunits.

    Dwivedi Y, Rizavi HS, Shukla PK, Lyons J, Faludi G, Palkovits M, Sarosi A, Conley RR, Roberts RC, Tamminga CA and Pandey GN

    Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor Street, Chicago, IL 60612, USA.

    Background: We recently reported reduced [3H]cyclic adenosine monophosphate binding and catalytic activity of protein kinase A in prefrontal cortex of depressed suicide victims. Here we examined the molecular basis of these alterations and whether these findings can be replicated in another cohort.

    Methods: Prefrontal cortex from depressed suicide victims and nonpsychiatric controls were obtained from the Lenhossek Human Brain Program, Budapest and the Maryland Brain Collection Program. [3H]cyclic adenosine monophosphate binding and protein kinase A activity were determined by radioligand binding and enzymatic assay, respectively. Expression of catalytic and regulatory subunits was determined by quantitative reverse transcription polymerase chain reaction and Western blot, respectively.

    Results: [3H]cyclic adenosine monophosphate binding and total and endogenous protein kinase A activity were significantly decreased in membrane and cytosol fractions of prefrontal cortex of depressed suicide victims from the Budapest cohort, with a similar magnitude (33%-40% reduction) as reported for the Maryland cohort. In both cohorts, selective reduction (36%-41%) in mRNA and protein expression of the regulatory RIIbeta and the catalytic Cbeta was observed.

    Conclusions: Our results suggest abnormalities in [3H]cyclic adenosine monophosphate binding and catalytic activity kinase A in brain of depressed suicide victims, which could be due to reduced expression of RIIbeta and Cbeta. These abnormalities in PKA may be critical in the pathophysiology of depression.

    Funded by: NIMH NIH HHS: K01 MH 01836, R01 MH 48153

    Biological psychiatry 2004;55;3;234-43

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

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

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

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

    Nature genetics 2004;36;1;40-5

  • 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

  • Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    Cartier C, Hemonnot B, Gay B, Bardy M, Sanchiz C, Devaux C and Briant L

    Laboratoire Infections Rétrovirales et Signalisation Cellulaire, Centre National de la Recherche Scientifique, UMR 5121-UM1, Institut de Biologie, CS 89508, 34960 Montpellier Cedex 2, France.

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

    The Journal of biological chemistry 2003;278;37;35211-9

  • Mutants of protein kinase A that mimic the ATP-binding site of protein kinase B (AKT).

    Gassel M, Breitenlechner CB, Rüger P, Jucknischke U, Schneider T, Huber R, Bossemeyer D and Engh RA

    German Cancer Research Center (DKFZ), Division of Pathochemistry, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

    The mutation of well behaved enzymes in order to simulate less manageable cognates is the obvious approach to study specific features of the recalcitrant target. Accordingly, the prototypical protein kinase PKA serves as a model for many kinases, including the closely related PKB, an AGC family protein kinase now implicated as oncogenic in several cancers. Two residues that differ between the alpha isoforms of PKA and PKB at the adenine-binding site generate differing shapes of the binding surface and are likely to play a role in ligand selectivity. As the corresponding mutations in PKA, V123A would enlarge the adenine pocket, while L173M would alter both the shape and its electronic character of the adenine-binding surface. We have determined the structures of the corresponding double mutant (PKAB2: PKAalpha V123A, L173M) in apo and MgATP-bound states, and observed structural alterations of a residue not previously involved in ATP-binding interactions: the side-chain of Q181, which in native PKA points away from the ATP-binding site, adopts in apo double mutant protein a new rotamer conformation, which places the polar groups at the hinge region in the ATP pocket. MgATP binding forces Q181 back to the position seen in native PKA. The crystal structure shows that ATP binding geometry is identical with that in native PKA but in this case was determined under conditions with only a single Mg ion ligand. Surface plasmon resonance spectroscopy studies show that significant energy is required for this ligand-induced transition. An additional PKA/PKB mutation, Q181K, corrects the defect, as shown both by the crystal structure of triple mutant PKAB3 (PKAalpha V123A, L173M, Q181K) and by surface plasmon resonance spectroscopy binding studies with ATP and three isoquinoline inhibitors. Thus, the triple mutant serves well as an easily crystallizable model for PKB inhibitor interactions. Further, the phenomenon of Q181 shows how crystallographic analysis should accompany mutant studies to monitor possible spurious structural effects.

    Journal of molecular biology 2003;329;5;1021-34

  • A humble hexose monophosphate pathway metabolite regulates short- and long-term control of lipogenesis.

    Veech RL

    Laboratory of Metabolism and Molecular Biology, Department of Health and Human Services, National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, 12501 Washington Avenue, Rockville, MD 20852, USA. rveech@mail.nih.gov

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;10;5578-80

  • PKA phosphorylates the p75 receptor and regulates its localization to lipid rafts.

    Higuchi H, Yamashita T, Yoshikawa H and Tohyama M

    Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-087, Japan.

    Although a large number of studies have been carried out on the diverse effects mediated by the common neurotrophin receptor p75(NTR), little is known about the molecular mechanisms by which p75(NTR) initiates intracellular signal transduction. We identified a variant of the beta catalytic subunit of cAMP-dependent protein kinase (PKACbeta) as a p75(NTR)-interacting protein, which phosphorylates p75(NTR) at Ser304. Intracellular cAMP in cerebellar neurons was accumulated transiently by ligand binding to p75(NTR). Activation of cAMP-PKA is required for translocation of p75(NTR) to lipid rafts, and for biochemical and biological activities of p75(NTR), such as inactivation of Rho and the neurite outgrowth. Proper recruitment of activated p75(NTR) to lipid rafts, structures that represent specialized signaling organelles, is of fundamental importance in determining p75(NTR) bioactivity.

    The EMBO journal 2003;22;8;1790-800

  • 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

  • c-MYC activates protein kinase A (PKA) by direct transcriptional activation of the PKA catalytic subunit beta (PKA-Cbeta) gene.

    Wu KJ, Mattioli M, Morse HC and Dalla-Favera R

    Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA.

    The c-MYC proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and differentiation and broadly implicated in tumorigenesis. Understanding the function of c-MYC and its role in cancer depends upon the identification of c-MYC target genes. Here we show that c-MYC induces the activity of Protein Kinase A (PKA), a key effector of cAMP-mediated signal transduction, by inducing the transcription of the gene encoding the PKA catalytic subunit beta (PKA-Cbeta). c-MYC-mediated induction of PKA-Cbeta gene transcription occurs in multiple tissues, is independent of cell proliferation and is mediated by direct binding of c-MYC to the PKA-Cbeta gene promoter sequences. Constitutive expression of PKA-Cbeta in Rat1A cells induces their transformation, and c-MYC-induced transformation can be reverted by pharmacological inhibition of PKA, suggesting that up-regulation of PKA is critical for c-MYC-associated tumorigenesis. These results indicate that, by activating PKA, c-MYC can provide endogenous activation of the cAMP signal transduction pathway independently of extracellular signals.

    Funded by: NCI NIH HHS: CA 37165

    Oncogene 2002;21;51;7872-82

  • Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

    Ørstavik S, Reinton N, Frengen E, Langeland BT, Jahnsen T and Skålhegg BS

    Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, Blindern, N-0316 Oslo, Norway.

    Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.

    European journal of biochemistry 2001;268;19;5066-73

  • The effects of aging on gene expression in the hypothalamus and cortex of mice.

    Jiang CH, Tsien JZ, Schultz PG and Hu Y

    Genomics Institute of the Novartis Research Foundation, 3115 Merryfield Row, San Diego, CA 92121, USA.

    A better understanding of the molecular effects of aging in the brain may help to reveal important aspects of organismal aging, as well as processes that lead to age-related brain dysfunction. In this study, we have examined differences in gene expression in the hypothalamus and cortex of young and aged mice by using high-density oligonucleotide arrays. A number of key genes involved in neuronal structure and signaling are differentially expressed in both the aged hypothalamus and cortex, including synaptotagmin I, cAMP-dependent protein kinase C beta, apolipoprotein E, protein phosphatase 2A, and prostaglandin D. Misregulation of these proteins may contribute to age-related memory deficits and neurodegenerative diseases. In addition, many proteases that play essential roles in regulating neuropeptide metabolism, amyloid precursor protein processing, and neuronal apoptosis are up-regulated in the aged brain and likely contribute significantly to brain aging. Finally, a subset of these genes whose expression is affected by aging are oppositely affected by exposure of mice to an enriched environment, suggesting that these genes may play important roles in learning and memory.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;4;1930-4

  • DNA cloning using in vitro site-specific recombination.

    Hartley JL, Temple GF and Brasch MA

    Life Technologies, Inc., Rockville, Maryland 20850, USA. jhartley@lifetech.com

    As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

    Genome research 2000;10;11;1788-95

  • 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

  • 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

  • The cAMP-dependent protein kinase A and protein kinase C-beta pathways synergistically interact to activate HIV-1 transcription in latently infected cells of monocyte/macrophage lineage.

    Rabbi MF, al-Harthi L, Saifuddin M and Roebuck KA

    Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA.

    The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathways. We demonstrate that the cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signaling pathways synergize to increase HIV-1 LTR-mediated transcription and viral replication in a latently infected promonocytic cell line (U1). The LTR-mediated synergy induced by cholera toxin (Ctx), a potent activator of the cAMP-dependent PKA pathway, and the PKC activator phorbol 12-myristate 13-acetate (PMA) was abrogated by a PKC-beta-specific inhibitor (LY333531). In contrast, the LTR-mediated synergy induced by Ctx and TNF alpha was not affected by LY333531. The synergy induced by Ctx and TNF alpha was also abrogated by mutation of the cAMP-responsive downstream sequence elements (DSE) in the 5' untranslated leader region, whereas the DSE mutations did not affect the synergy induced by Ctx and PMA. These distinctions indicate that Ctx cooperates differently with TNF alpha and PMA to activate the HIV-1 LTR. Ctx and PMA synergistically activated AP-1- and NF-kappa B-dependent transcription, even though no cooperative binding of AP-1 or NF-kappa B was observed in gel shift assays. An extensive mutational analysis of the HIV-1 LTR that included the NF-kappa B and AP-1 binding sites revealed no distinct cis-acting element or region within the HIV-1 LTR that was required for the transcriptional synergy. Ctx and PMA also synergistically interact to activate the HTLV-1 LTR. These results indicate that the transcriptional synergy elicited by Ctx and PMA targets multiple functional elements and promoters, requires a cooperative interaction between the PKA and PKC-beta pathways, and differs mechanistically from the transcriptional synergy induced by Ctx and TNF alpha.

    Virology 1998;245;2;257-69

  • 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

  • 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

  • The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo.

    Gamm DM, Baude EJ and Uhler MD

    Department of Biological Chemistry, and the Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.

    Two isoforms of the catalytic subunit of cAMP-dependent protein kinase, Calpha and Cbeta1, are known to be widely expressed in mammals. Although much is known about the structure and function of Calpha, few studies have addressed the possibility of a distinct role for the Cbeta proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. Cbeta1 demonstrated higher Km values for some peptide substrates than did Calpha, but Cbeta1 was insensitive to substrate inhibition, a phenomenon that was observed with Calpha at substrate concentrations above 100 microM. Calpha and Cbeta1 displayed distinct IC50 values for the alpha and beta isoforms of the protein kinase inhibitor, protein kinase inhibitorpeptide, and the type IIalpha regulatory subunit (RIIalpha). Of particular interest, purified type II holoenzyme containing Cbeta1 exhibited a 5-fold lower Ka value for cAMP (13 nM) than did type II holoenzyme containing Calpha (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RIIalpha2Cbeta12 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RIIalpha2Calpha2 holoenzyme. These results provide evidence that type II holoenzyme formed with Cbeta1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.

    Funded by: NIGMS NIH HHS: GM38788

    The Journal of biological chemistry 1996;271;26;15736-42

  • 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

  • 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

  • Evidence for two additional isoforms of the endogenous protein kinase inhibitor of cAMP-dependent protein kinase in mouse.

    Scarpetta MA and Uhler MD

    Mental Health Research Institute, University of Michigan, Ann Arbor 48109.

    Oligonucleotides derived from the previously published rat testicular protein kinase inhibitor (PKI) sequence (Van Patten, S. M., Ng, D. C., Th'ng, J. P. H., Angelos, K. L., Smith, A. J., and Walsh, D. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5383-5387) were used to isolate a DNA fragment coding for a mouse homologue of the rat testicular PKI. This DNA fragment was then used to screen a mouse brain cDNA library, and two cDNA clones related to the testicular PKI (PKI beta) were isolated. Sequencing and comparison showed that the two cDNAs differ only in the presence of a 105-base pair insert, which results in an amino-terminal extension of the predicted PKI beta 2 protein by 20 residues relative to the PKI beta 1 protein. By using the appropriate primers, PCR was used to amplify specific regions of both of these clones from mouse brain cDNA. When both clones were expressed in vitro, the mRNA for PKI beta 2 produced a protein product that was larger and much more effectively translated, suggesting a functional role for the inserted sequence. Both isoforms were transiently expressed in COS-1 cells to evaluate their ability to inhibit the catalytic subunit of PKA in vivo. Extracts from cells expressing the PKI beta 2 isoform showed greater inhibition of catalytic subunit kinase activity than extracts expressing the PKI beta 1 isoform. Northern blot analysis of poly(A)+ RNA from various mouse tissues showed the presence of transcripts of 1.8 kilobases related to these cDNAs. Analysis of brain RNA from several species indicated that expression of these PKI mRNAs is evolutionarily conserved. Together with previous studies, these results indicate the presence of at least three PKI proteins in mouse. The skeletal muscle isoform has been designated PKI alpha, while the testicular isoform is PKI beta. We propose to designate the two testicular isoforms described here PKI beta 1 and PKI beta 2.

    Funded by: NIGMS NIH HHS: GM 38788; NIMH NIH HHS: MH15794

    The Journal of biological chemistry 1993;268;15;10927-31

  • Assignment of the gene encoding the catalytic subunit C beta of cAMP-dependent protein kinase to the p36 band on chromosome 1.

    Simard J, Bérubé D, Sandberg M, Grzeschik KH, Gagné R, Hansson V and Jahnsen T

    MRC Group in Molecular Endocrinology, CHUL Research Center and Laval University, Québec, Canada.

    A cDNA for the human catalytic subunit (C beta) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C beta as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kb XbaI fragment, which hybridized with the C beta probe, was concordant with the presence of human chromosome 1. In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C beta gene of PKA to the p36 band on chromosome 1.

    Human genetics 1992;88;6;653-7

  • 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

  • Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis--representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase.

    Beebe SJ, Oyen O, Sandberg M, Frøysa A, Hansson V and Jahnsen T

    Institute of Pathology, Rikshospitalet, Oslo, Norway.

    Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.

    Molecular endocrinology (Baltimore, Md.) 1990;4;3;465-75

  • 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

Gene lists (7)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000034 G2C Homo sapiens Pocklington H3 Human orthologues of cluster 3 (mouse) from Pocklington et al (2006) 30
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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