G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1)
G00000176 (Mus musculus)

Databases (7)

ENSG00000108946 (Ensembl human gene)
5573 (Entrez Gene)
306 (G2Cdb plasticity & disease)
PRKAR1A (GeneCards)
188830 (OMIM)
Marker Symbol
HGNC:9388 (HGNC)
Protein Sequence
P10644 (UniProt)

Literature (118)

Pubmed - other

  • Leukemic transformation by the APL fusion protein PRKAR1A-RAR{alpha} critically depends on recruitment of RXR{alpha}.

    Qiu JJ, Lu X, Zeisig BB, Ma Z, Cai X, Chen S, Gronemeyer H, Tweardy DJ, So CW and Dong S

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

    PRKAR1A (R1A)-retinoic acid receptor-alpha (R1A-RARalpha) is the sixth RARalpha-containing fusion protein in acute promyelocytic leukemia (APL). Using the murine bone-marrow retroviral transduction/transformation assay, we showed that R1A-RARalpha fusion protein could transform bone-marrow progenitor/stem cells. In gel-shift assays, R1A-RARalpha was able to bind to a panel of retinoic acid response elements both as a homodimer and as a heterodimer with RXRalpha, and demonstrated distinct DNA-binding characteristics compared with wild-type RARalpha/RXRalpha or other X-RARalpha chimeric proteins. The ratio of R1A-RARalpha to RXRalpha proteins affected the retinoic acid response element interaction pattern of R1A-RARalpha/RXRalpha complexes. Studies comparing R1A-RARalpha with R1A-RARalpha(DeltaRIIa) demonstrated that the RIIa protein interaction domain located within R1A was responsible for R1A-RARalpha homodimeric DNA binding and interaction with wild-type R1A protein. However, the RIIa domain was not required for R1A-RARalpha-mediated transformation because its deletion in R1A-RARalpha(DeltaRIIa) did not compromise its transformation capability. In contrast, introduction of point mutations within the RARalpha portion of either R1A-RARalpha or R1A-RARalpha(DeltaRIIa), previously demonstrated to eliminate RXRalpha interaction or treatment of transduced cells with RXRalpha shRNA or a RXRalpha agonist, reduced transformation capability. Thus, leukemic transformation by APL fusion protein PRKAR1A-RARalpha is critically dependent on RXRalpha, which suggests RXRalpha is a promising target for APL.

    Funded by: Medical Research Council: G0800892

    Blood 2010;115;3;643-52

  • Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278).

    Yang WL, Ravatn R, Kudoh K, Alabanza L and Chin KV

    Long Island Jewish Medical Center, North Shore University Hospital, Manhasset, NY 11030, USA.

    The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R(2)C(2). The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RIalpha, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RIalpha, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RIalpha subunit of PKA may have functions independent of the kinase. We show here that the RIalpha subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RIalpha results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RIalpha and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RIalpha modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RIalpha with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

    Funded by: NCI NIH HHS: R01 CA102204, R01 CA102204-01A2

    Biochemical and biophysical research communications 2010;391;3;1318-23

  • Association of the M1V PRKAR1A mutation with primary pigmented nodular adrenocortical disease in two large families.

    Pereira AM, Hes FJ, Horvath A, Woortman S, Greene E, Bimpaki E, Alatsatianos A, Boikos S, Smit JW, Romijn JA, Nesterova M and Stratakis CA

    Department of Endocrinology and Metabolism and Center for Human, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

    Background: Carney complex (CNC) is a familial multiple neoplasia syndrome frequently associated with primary pigmented nodular adrenocortical disease (PPNAD), a bilateral form of micronodular adrenal hyperplasia that leads to Cushing's syndrome (CS). Germline PRKAR1A mutations cause CNC and only rarely isolated PPNAD.

    PRKAR1A mutation analysis in two large families with CS and no other CNC manifestations demonstrated a M1V germline mutation; a total of 21 asymptomatic individuals were screened, and mutation carriers were evaluated for CNC. The mutation was expressed in vitro and functionally tested for its effects on protein kinase A function.

    Results: Presymptomatic testing identified five first-degree relatives who were M1V carriers and who were all diagnosed with subclinical, mild CS at ages ranging from 20-56 yr. There were no other signs of CNC. In a cell-free system, we detected a shorter compared with the wild-type type 1alpha regulatory subunit of protein kinase A (PRKAR1A) protein (43 kDa). This was not identified in cell lines from the patients or in transfection experiments in HEK293 cells that showed no detectable PRKAR1A protein from the M1V-bearing constructs. In these cells, the mutant mRNA was expressed in a 1:1 ratio.

    Conclusion: In two large families, the M1V PRKAR1A mutation resulted in a PPNAD-only phenotype with significant variability both in terms of age of onset and clinical severity. Expression studies showed a unique effect of this sequence change. This study has implications for genetic counseling of carriers of this PRKAR1A mutation and patients with CNC and PPNAD and for the study of PRKAR1A-related tumorigenesis.

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

    The Journal of clinical endocrinology and metabolism 2010;95;1;338-42

  • The metastasis efficiency modifier ribosomal RNA processing 1 homolog B (RRP1B) is a chromatin-associated factor.

    Crawford NP, Yang H, Mattaini KR and Hunter KW

    Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

    There is accumulating evidence for a role of germ line variation in breast cancer metastasis. We have recently identified a novel metastasis susceptibility gene, Rrp1b (ribosomal RNA processing 1 homolog B). Overexpression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in breast cancer. Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicted the outcome in three of four publicly available breast carcinoma gene expression data sets. In addition, we provide insights into the mechanism of RRP1B. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, including histone H1X, poly(ADP-ribose) polymerase 1, TRIM28 (tripartite motif-containing 28), and CSDA (cold shock domain protein A). Co-immunofluorescence and co-immunoprecipitation confirmed these interactions and also interactions with heterochromatin protein-1alpha and acetyl-histone H4 lysine 5. Finally, we investigated the effects of ectopic expression of an RRP1B allelic variant previously associated with improved survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wild type RRP1B in HeLa cells, the variant RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure.

    Funded by: Intramural NIH HHS

    The Journal of biological chemistry 2009;284;42;28660-73

  • 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

  • Inactivation of the Carney complex gene 1 (protein kinase A regulatory subunit 1A) inhibits SMAD3 expression and TGF beta-stimulated apoptosis in adrenocortical cells.

    Ragazzon B, Cazabat L, Rizk-Rabin M, Assie G, Groussin L, Fierrard H, Perlemoine K, Martinez A and Bertherat J

    Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique (UMR 8104), Paris, France.

    The cyclic AMP signaling pathway can be altered at multiple levels in endocrine tumors. Its central component is the protein kinase A (PKA). Carney complex (CNC) is a hereditary multiple neoplasia syndrome resulting from inactivating mutations of the gene encoding the PKA type I alpha regulatory subunit (PRKAR1A). Primary pigmented nodular adrenocortical disease is the most frequent endocrine tumor of CNC. Transforming growth factor beta (TGFbeta) regulates adrenal cortex physiology and signals through SMAD2/3. We used an interference approach to test the effects of PRKAR1A inactivation on PKA and TGFbeta pathways and on apoptosis in adrenocortical cells. PRKAR1A silencing stimulates PKA activity and increases transcriptional activity of a PKA reporter construct and expression of the endogenous PKA target, NR4A2, under basal conditions or after forskolin stimulation. PRKAR1A inactivation also decreased SMAD3 mRNA and protein levels via PKA, altering the cellular response to TGFbeta. SMAD3 expression was also inhibited by adrenocorticorticotropic hormone in the mouse adrenal gland and by forskolin in H295R cells. TGFbeta stimulates apoptosis in H295R cells, and this effect was counteracted by PRKAR1A inactivation. PRKAR1A silencing decreased the percentage of apoptotic cells and the cleavage of apoptosis mediators [caspase-3, poly(ADP-ribose) polymerase, and lamin A/C]. Inactivating mutations of PRKAR1A observed in adrenocortical tumors alter SMAD3, leading to resistance to TGFbeta-induced apoptosis. This cross-talk between the PKA and the TGFbeta signaling pathways reveals a new mechanism of endocrine tumorigenesis.

    Cancer research 2009;69;18;7278-84

  • The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors.

    Landa I, Ruiz-Llorente S, Montero-Conde C, Inglada-Pérez L, Schiavi F, Leskelä S, Pita G, Milne R, Maravall J, Ramos I, Andía V, Rodríguez-Poyo P, Jara-Albarrán A, Meoro A, del Peso C, Arribas L, Iglesias P, Caballero J, Serrano J, Picó A, Pomares F, Giménez G, López-Mondéjar P, Castello R, Merante-Boschin I, Pelizzo MR, Mauricio D, Opocher G, Rodríguez-Antona C, González-Neira A, Matías-Guiu X, Santisteban P and Robledo M

    Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

    In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

    PLoS genetics 2009;5;9;e1000637

  • Regulatory subunit I-controlled protein kinase A activity is required for apical bile canalicular lumen development in hepatocytes.

    Wojtal KA, Diskar M, Herberg FW, Hoekstra D and van Ijzendoorn SC

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

    Signaling via cAMP plays an important role in apical cell surface dynamics in epithelial cells. In hepatocytes, elevated levels of cAMP as well as extracellular oncostatin M stimulate apical lumen development in a manner that depends on protein kinase A (PKA) activity. However, neither the identity of PKA isoforms involved nor the mechanisms of the cross-talk between oncostatin M and cAMP/PKA signaling pathways have been elucidated. Here we demonstrate that oncostatin M and PKA signaling converge at the level of the PKA holoenzyme downstream of oncostatin M-stimulated MAPK activation. Experiments were performed with chemically modified cAMP analogues that preferentially target regulatory subunit (R) I or RII holoenzymes, respectively, in hepatocytes. The data suggest that the dissociation of RI- but not RII-containing holoenzymes, as well as catalytic activity of PKA, is required for apical lumen development in response to elevated levels of cAMP and oncostatin M. However, oncostatin M signaling does not stimulate PKA holoenzyme dissociation in living cells. Based on pharmacological and cell biological studies, it is concluded that RI-controlled PKA activity is essential for cAMP- and oncostatin M-stimulated development of apical bile canalicular lumens.

    The Journal of biological chemistry 2009;284;31;20773-80

  • Abnormalities of cAMP signaling are present in adrenocortical lesions associated with ACTH-independent Cushing syndrome despite the absence of mutations in known genes.

    Bimpaki EI, Nesterova M and Stratakis CA

    Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), NIH, Building 10, CRC (East Laboratories), Room 1-3330, 10 Center Drive, MSC1103, Bethesda, Maryland 20892, USA.

    Context: Bilateral adrenal hyperplasias (BAHs) may be caused by mutations of genes that code for molecules that participate in cAMP signaling. Little is known about cAMP signaling in adrenal lesions associated with ACTH-independent Cushing syndrome (AICS) that do not harbor mutations in known genes.

    Objective: We assessed the cAMP-signaling pathway by enzymatic and molecular studies.

    Design: Samples from 27 patients (ages 5-60 years) were studied and compared with normal adrenocortical tissue (n=4) and aldosterone-producing adenomas (APA, n=5). All samples were sequenced for GNAS, PRKAR1A, PDE11A, and PDE8B sequencing defects. cAMP levels and binding, protein kinase A, and phosphodiesterase (PDE) activities were assayed. Immunohistochemistry was used for certain studies and the phosphorylation status of CREB was studied.

    Patients: A total of 36 samples from patients were used.

    Results: Cortisol-producing adenomas (CPAs) and other lesions that were GNAS, PRKAR1A, PDE11A, and PDE8B gene mutation-negative were compared with PRKAR1A mutation-positive lesions, normal tissue, and APAs; abnormalities of the cAMP-signaling pathway were found in both BAHs and CPAs. Interestingly, mutation-negative CPAs had significantly decreased PDE activity.

    Conclusion: Lesions of the adrenal associated with AICS, independently of their GNAS, PRKAR1A, PDE11A, and PDE8B mutation status, have functional abnormalities of cAMP signaling. It is probable that epigenetic events or additional defects of genes involved in this pathway are responsible for this phenomenon.

    Funded by: Intramural NIH HHS: ZIA HD000642-13; NICHD NIH HHS: Z01 HD000642, Z01-HD-000642-04

    European journal of endocrinology 2009;161;1;153-61

  • Mutations in regulatory subunit type 1A of cyclic adenosine 5'-monophosphate-dependent protein kinase (PRKAR1A): phenotype analysis in 353 patients and 80 different genotypes.

    Bertherat J, Horvath A, Groussin L, Grabar S, Boikos S, Cazabat L, Libe R, René-Corail F, Stergiopoulos S, Bourdeau I, Bei T, Clauser E, Calender A, Kirschner LS, Bertagna X, Carney JA and Stratakis CA

    Institut National de la Santé et de la Recherche Médicale Unit 567, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Institut Cochin, Endocrinology, Metabolism and Cancer Department, Paris 75014, France.

    Background: The "complex of myxomas, spotty skin pigmentation, and endocrine overactivity," or "Carney complex" (CNC), is caused by inactivating mutations of the regulatory subunit type 1A of the cAMP-dependent protein kinase (PRKAR1A) gene and as yet unknown defect(s) in other gene(s). Delineation of a genotype-phenotype correlation for CNC patients is essential for understanding PRKAR1A function and providing counseling and preventive care.

    Methods: A transatlantic consortium studied the molecular genotype and clinical phenotype of 353 patients (221 females and 132 males, age 34 +/- 19 yr) who carried a germline PRKAR1A mutation or were diagnosed with CNC and/or primary pigmented nodular adrenocortical disease.

    Results: A total of 258 patients (73%) carried 80 different PRKAR1A mutations; 114 (62%) of the index cases had a PRKAR1A mutation. Most PRKAR1A mutations (82%) led to lack of detectable mutant protein (nonexpressed mutations) because of nonsense mRNA mediated decay. Patients with a PRKAR1A mutation were more likely to have pigmented skin lesions, myxomas, and thyroid and gonadal tumors; they also presented earlier with these tumors. Primary pigmented nodular adrenocortical disease occurred earlier, was more frequent in females, and was the only manifestation of CNC with a gender predilection. Mutations located in exons were more often associated with acromegaly, myxomas, lentigines, and schwannomas, whereas the frequent c.491-492delTG mutation was commonly associated with lentigines, cardiac myxomas, and thyroid tumors. Overall, nonexpressed PRKAR1A mutations were associated with less severe disease.

    Conclusion: CNC is genetically and clinically heterogeneous. Certain tumors are more frequent, with specific mutations providing some genotype-phenotype correlation for PRKAR1A mutations.

    Funded by: Intramural NIH HHS; NICHD NIH HHS: Z01 HD000642

    The Journal of clinical endocrinology and metabolism 2009;94;6;2085-91

  • Analysis of GNAS1 and PRKAR1A gene mutations in human cardiac myxomas not associated with multiple endocrine disorders.

    Mantovani G, Bondioni S, Corbetta S, Menicanti L, Rubino B, Peverelli E, Labarile P, Dall'Asta C, Ambrosi B, Beck-Peccoz P, Lania AG and Spada A

    Endocrine Unit, Department of Medical Sciences, Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena IRCCS, 20122 Milan, Italy. giovanna.mantovani@unimi.it

    Cardiac myxomas are rare tumors that usually occur as sporadic lesions or,more rarely, in the familial form,mostly in the context of Carney complex (CNC). The molecular basis for the development of cardiac myxomas is unclear. However, somatic activating mutations in the GNAS1 gene (the gsp oncogene) are detected in the myocardium ofMcCune-Albright syndrome patients while germ-line mutations in the PRKAR1A gene are associated with CNC and familial myxomas. We investigated the presence of activating missense mutations in the GNAS1 gene as well as of inactivating mutations in PRKAR1A in 29 sporadically occurring cardiac myxomas. No gsp and no PRKAR1A mutations were found by direct sequencing of PCR products amplified from tumoral DNA. This is the first study including a large series of sporadic, isolated cardiac myxomas and showing that these cardiac neoplasms do not share the same mutations found in familial forms.

    Journal of endocrinological investigation 2009;32;6;501-4

  • An empirical framework for binary interactome mapping.

    Venkatesan K, Rual JF, Vazquez A, Stelzl U, Lemmens I, Hirozane-Kishikawa T, Hao T, Zenkner M, Xin X, Goh KI, Yildirim MA, Simonis N, Heinzmann K, Gebreab F, Sahalie JM, Cevik S, Simon C, de Smet AS, Dann E, Smolyar A, Vinayagam A, Yu H, Szeto D, Borick H, Dricot A, Klitgord N, Murray RR, Lin C, Lalowski M, Timm J, Rau K, Boone C, Braun P, Cusick ME, Roth FP, Hill DE, Tavernier J, Wanker EE, Barabási AL and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, 1 Jimmy Fund Way, Boston, MA 02115, USA.

    Several attempts have been made to systematically map protein-protein interaction, or 'interactome', networks. However, it remains difficult to assess the quality and coverage of existing data sets. Here we describe a framework that uses an empirically-based approach to rigorously dissect quality parameters of currently available human interactome maps. Our results indicate that high-throughput yeast two-hybrid (HT-Y2H) interactions for human proteins are more precise than literature-curated interactions supported by a single publication, suggesting that HT-Y2H is suitable to map a significant portion of the human interactome. We estimate that the human interactome contains approximately 130,000 binary interactions, most of which remain to be mapped. Similar to estimates of DNA sequence data quality and genome size early in the Human Genome Project, estimates of protein interaction data quality and interactome size are crucial to establish the magnitude of the task of comprehensive human interactome mapping and to elucidate a path toward this goal.

    Funded by: NCI NIH HHS: 5U01 CA 105423, 5U54 CA 112952, T32 CA 09361, T32 CA009361, U01 CA105423, U54 CA112952, U56 CA 113004, U56 CA113004; NHGRI NIH HHS: 2R01 HG 001715, 5P50 HG 004233, P50 HG004233, P50 HG004233-03, R01 HG001715, R01 HG001715-12; NIAID NIH HHS: U01 AI 070499-01

    Nature methods 2009;6;1;83-90

  • Protein kinase A-mediated gating of neuregulin-dependent ErbB2-ErbB3 activation underlies the synergistic action of cAMP on Schwann cell proliferation.

    Monje PV, Athauda G and Wood PM

    Miami Project to Cure Paralysis, Miami, FL 33136, USA. MPaula@miamiproject.med.miami.edu

    In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.

    Funded by: NINDS NIH HHS: NS009923

    The Journal of biological chemistry 2008;283;49;34087-100

  • A novel PRKAR1A mutation associated with hepatocellular carcinoma in a young patient and a variable Carney complex phenotype in affected subjects in older generations.

    Gennari M, Stratakis CA, Hovarth A, Pirazzoli P and Cicognani A

    Paediatric Endocrinology, S. Orsola Hospital-University of Bologna, Bologna, Italy. monia.gennari2@unibo.it

    Context: Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia syndrome (OMIM 160980). About 70% of cases are familiar; most have mutations of the PRKAR1A gene on chromosome 17q22-24. There is little phenotype-genotype correlation known to date.

    Objective: To study the genotype-phenotype correlation in a family with newly diagnosed CNC and three generations of subjects bearing the same PRKAR1A mutation. The proband was diagnosed with hepatocellular carcinoma, a tumour that appears to be associated with CNC.

    Design: The study consisted of clinical and genetic analysis of a total of 10 individuals belonging to a large Italian family.

    Patients: The index case was referred for PRKAR1A gene mutation analysis because he met the diagnostic criteria for a clinical diagnosis of CNC.

    Results: The PRKAR1A-inactivating mutation c.502 +1G > A in the intron 5 splice-donor site was detected after bidirectional sequencing of germline DNA. The mutation causes a frameshift in the transcribed sequence and a nonsense mRNA that was shown to be degraded; this leads to PRKAR1A haploinsufficiency in all tissues. All available relatives were screened first by DNA testing and, if the latter was positive, by clinical, biochemical and imaging means.

    Conclusions: A novel PRKAR1A mutation with an apparently low penetrance and variable expression is reported; the same mutation is also associated with a hepatocellular carcinoma. This is the first time a PRKAR1A mutation is reported in individuals who were diagnosed with CNC after retrospective family screening and following the identification of a proband; the finding has implications for genetic counselling on PRKAR1A and/or CNC.

    Funded by: Intramural NIH HHS: ZIA HD000642-13

    Clinical endocrinology 2008;69;5;751-5

  • Case report of familial Carney complex due to novel frameshift mutation c.597del C (p.Phe200LeufsX6) in PRKAR1A.

    Sasaki A, Horikawa Y, Suwa T, Enya M, Kawachi S and Takeda J

    Department of Diabetes and Endocrinology, Division of Molecule and Structure, Gifu University School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan.

    Carney complex is an autosomal dominantly inherited disease characterized by skin pigmentation, myxoma, primary pigmented nodular adrenocortical disease (PPNAD), and acromegaly. However, only a few incidences of PPNAD combined with acromegaly are observed in patients. The type 1alpha regulatory subunit of cAMP-dependent protein kinase (PRKAR1A) has been identified in patients as a causative gene for Carney complex by a positional cloning approach. Here, we report a female patient diagnosed with Cushing's syndrome and a GH-producing pituitary adenoma without otherwise evident acromegaly that could be diagnosed only by specialized endocrinological tests. Based on family history of acromegaly (mother and sister) and the fact that the combination of both diseases is very rare, genetic diagnosis involving Carney complex was considered to be appropriate. The 10 exons and flanking regions of PRKAR1A were screened for mutations by direct DNA sequencing. The patient and her mother and sister were found to have the same, novel frameshift mutation resulting from a single base deletion in exon 6 coding cAMP-binding domain A, denoted c.597delC in PRKAR1A. This single base deletion generated an immature stop codon at the sixth codon (p.Phe200LeufsX6). Even family members with the same mutation can show distinct phenotypes, suggesting that Carney complex is a multifactorial disorder comprising various genetic and environmental factors. Genetic diagnosis makes it possible to prepare more effective therapeutic strategies for patients and gene carriers and to avoid unnecessary tests for non-carriers in the family of the patient.

    Molecular genetics and metabolism 2008;95;3;182-7

  • Wnt/beta-catenin and 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors.

    Gaujoux S, Tissier F, Groussin L, Libé R, Ragazzon B, Launay P, Audebourg A, Dousset B, Bertagna X and Bertherat J

    Service des Maladies Endocriniennes et Métaboliques, Hôpital Cochin, 75014 Paris, France.

    Background: The Wnt/beta-catenin and cAMP signaling pathways play an important role in adrenal cortex tumorigenesis. Somatic activating mutations of the beta-catenin gene (CTNNB1) are the most frequent genetic defects identified both in adrenocortical adenomas (ACAs) and adrenocortical cancers (ACCs). PRKAR1A mutations leading to cAMP pathway dysregulation are observed in primary pigmented nodular adrenocortical diseases (PPNADs) and some sporadic ACAs.

    Objective: The objective of the investigation was to study Wnt/beta-catenin dysregulation in adrenocortical tumors (ACTs) with cAMP pathway genetic alteration and search for secondary CTNNB1 somatic mutations in heterogeneous tumors.

    Nine PPNADs, including five with macronodules, three ACAs with PRKAR1A somatic mutations, and one heterogeneous tumor with ACC developed within an ACA, were studied by immunohistochemistry and DNA sequencing.

    Results: beta-Catenin accumulation was observed in all PPNADs, ACAs with PRKAR1A mutations, and the ACC component of the heterogeneous tumor. CTNNB1 somatic activating mutations were found in the macronodule of two of the five macronodular PPNADs, in one ACA with a PRKAR1A somatic mutation, and in the malignant part of the heterogeneous ACT.

    Conclusions: The Wnt/beta-catenin pathway is activated in PPNADs and ACAs with PRKAR1A mutations, suggesting a cross talk between the cAMP and Wnt/beta-catenin pathways in ACT development. In addition, the occurrence as an additional hit of a CTNNB1 somatic mutation is associated with larger or more aggressive ACTs. This underlines the importance of the Wnt/beta-catenin pathway in adrenal cortex tumorigenesis and the importance of genetic accumulation in the progression of ACTs.

    The Journal of clinical endocrinology and metabolism 2008;93;10;4135-40

  • Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways.

    Torii K, Nishizawa K, Kawasaki A, Yamashita Y, Katada M, Ito M, Nishimoto I, Terashita K, Aiso S and Matsuoka M

    Noevir-Keio Research Laboratory, Noevir Co., Ltd., 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

    Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.

    Cellular signalling 2008;20;7;1256-66

  • Protein kinase A subunit expression is altered in Bloom syndrome fibroblasts and the BLM protein is increased in adrenocortical hyperplasias: inverse findings for BLM and PRKAR1A.

    Heyerdahl SL, Boikos S, Horvath A, Giatzakis C, Bossis I and Stratakis CA

    Section on Endocrinology & Genetics, Program on Developmental Endocrinology and Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition to tumors that is caused by germline mutations of the BLM gene, a RecQ helicase. Benign adrenocortical tumors display a degree of chromosomal instability that is more significant than benign tumors of other tissues. Cortisol-producing hyperplasias, such as primary pigmented nodular adrenocortical disease (PPNAD), which has been associated with protein kinase A (PKA) abnormalities and/or PRKAR1A mutations, also show genomic instability. Another RecQ helicase, WRN, directly interacts with the PRKAR1B subunit of PKA. In this study, we have investigated the PRKAR1A expression in primary human Bloom syndrome cell lines with known BLM mutations and examined the BLM gene expression in PPNAD and other adrenal tumor tissues. PRKAR1A and other protein kinase A (PKA) subunits were expressed in Bloom syndrome cells and their level of expression differed by subunit and cell type. Overall, fibroblasts exhibited a significant decrease in protein expression of all PKA subunits except for PRKAR1A, a pattern that has been associated with neoplastic transformation in several cell types. The BLM protein was upregulated in PPNAD and other hyperplasias, compared to samples from normal adrenals and normal cortex, as well as samples from cortisol- and aldosterone-producing adenomas (in which BLM was largely absent). These data reveal an inverse relationship between BLM and PRKAR1A: BLM deficiency is associated with a relative excess of PRKAR1A in fibroblasts compared to other PKA subunits; and PRKAR1A deficiency is associated with increased BLM protein in adrenal hyperplasias.

    Funded by: Intramural NIH HHS

    Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme 2008;40;6;391-7

  • Protein kinase A effects of an expressed PRKAR1A mutation associated with aggressive tumors.

    Meoli E, Bossis I, Cazabat L, Mavrakis M, Horvath A, Stergiopoulos S, Shiferaw ML, Fumey G, Perlemoine K, Muchow M, Robinson-White A, Weinberg F, Nesterova M, Patronas Y, Groussin L, Bertherat J and Stratakis CA

    Section on Endocrinology and Genetics, Program in Developmental Endocrinology and Genetics, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892, USA.

    Most PRKAR1A tumorigenic mutations lead to nonsense mRNA that is decayed; tumor formation has been associated with an increase in type II protein kinase A (PKA) subunits. The IVS6+1G>T PRKAR1A mutation leads to a protein lacking exon 6 sequences [R1 alpha Delta 184-236 (R1 alpha Delta 6)]. We compared in vitro R1 alpha Delta 6 with wild-type (wt) R1 alpha. We assessed PKA activity and subunit expression, phosphorylation of target molecules, and properties of wt-R1 alpha and mutant (mt) R1 alpha; we observed by confocal microscopy R1 alpha tagged with green fluorescent protein and its interactions with Cerulean-tagged catalytic subunit (C alpha). Introduction of the R1 alpha Delta 6 led to aberrant cellular morphology and higher PKA activity but no increase in type II PKA subunits. There was diffuse, cytoplasmic localization of R1 alpha protein in wt-R1 alpha- and R1 alpha Delta 6-transfected cells but the former also exhibited discrete aggregates of R1 alpha that bound C alpha; these were absent in R1 alpha Delta 6-transfected cells and did not bind C alpha at baseline or in response to cyclic AMP. Other changes induced by R1 alpha Delta 6 included decreased nuclear C alpha. We conclude that R1 alpha Delta 6 leads to increased PKA activity through the mt-R1 alpha decreased binding to C alpha and does not involve changes in other PKA subunits, suggesting that a switch to type II PKA activity is not necessary for increased kinase activity or tumorigenesis.

    Funded by: Intramural NIH HHS: ZIA HD000642-13; NICHD NIH HHS: Z01 HD000642

    Cancer research 2008;68;9;3133-41

  • In vitro functional studies of naturally occurring pathogenic PRKAR1A mutations that are not subject to nonsense mRNA decay.

    Greene EL, Horvath AD, Nesterova M, Giatzakis C, Bossis I and Stratakis CA

    Section on Endocrinology & Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Patients presenting with primary pigmented nodular adrenocortical disease (PPNAD), Carney complex (CNC), or sporadic tumors were previously found to carry germline mutations in the human type Ialpha regulatory subunit (RIalpha) of adenosine 3',5'-cyclic monophosphate (cyclic AMP [cAMP])-dependent protein kinase (PKA; PRKAR1A). Although about 90% of disease-causing PRKAR1A mutations lead to premature stop codon generation and subsequent degradation of the mutant message by nonsense-mediated mRNA decay (NMD), here we describe seven PRKAR1A mutations whose mRNAs do not seem to undergo NMD and instead result in an expressed mutant RIalpha protein. The expressed mutations (p.Ser9Asn, p.Glu60_Lys116del [Delta-exon 3], p.Arg74Cys, p.Arg146Ser, p.Asp183Tyr, p.Ala213Asp, and p.Gly289Trp) were spread over all the functional RIalpha domains, and all of them exhibited increased PKA activity, which we attribute to decreased binding to cAMP and/or the catalytic subunit. Our data further corroborate the previous finding that altered PRKAR1A function, not only haploinsufficiency, is enough to elevate PKA activity which is apparently associated with tumorigenesis in tissues affected by CNC. In some cases, as with the Delta-exon 3 mutation, we may even conclude that the presence of a mutant PRKAR1A protein may be more harmful than allelic loss.

    Funded by: Intramural NIH HHS

    Human mutation 2008;29;5;633-9

  • Targeted deletion of Prkar1a reveals a role for protein kinase A in mesenchymal-to-epithelial transition.

    Nadella KS, Jones GN, Trimboli A, Stratakis CA, Leone G and Kirschner LS

    Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, USA.

    Dysregulation of protein kinase A (PKA) activity, caused by loss of function mutations in PRKAR1A, is known to induce tumor formation in the inherited tumor syndrome Carney complex (CNC) and is also associated with sporadic tumors of the thyroid and adrenal. We have previously shown that Prkar1a(+/-) mice develop schwannomas reminiscent of those seen in CNC and that similar tumors are observed in tissue-specific knockouts (KO) of Prkar1a targeted to the neural crest. Within these tumors, we have previously described the presence of epithelial islands, although the nature of these structures was unclear. In this article, we report that these epithelial structures are derived from KO cells originating in the neural crest. Analysis of the mesenchymal marker vimentin revealed that this protein was markedly down-regulated not only from the epithelial islands, but also from the tumor as a whole, consistent with mesenchymal-to-epithelial transition (MET). In vitro, Prkar1a null primary mouse embryonic fibroblasts, which display constitutive PKA signaling, also showed evidence for MET, with a loss of vimentin and up-regulation of the epithelial marker E-cadherin. Reduction of vimentin protein occurred at the posttranslational level and was rescued by proteasomal inhibition. Finally, this down-regulation of vimentin was recapitulated in the adrenal nodules of CNC patients, confirming an unexpected and previously unrecognized role for PKA in MET.

    Funded by: Intramural NIH HHS: ZIA HD000642-13; NCI NIH HHS: CA112268-02, CA16058, P30 CA016058, R01 CA112268; NICHD NIH HHS: HD01323, K22 HD001323, Z01 HD000642

    Cancer research 2008;68;8;2671-7

  • High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells.

    Mantovani G, Bondioni S, Lania AG, Rodolfo M, Peverelli E, Polentarutti N, Veliz Rodriguez T, Ferrero S, Bosari S, Beck-Peccoz P and Spada A

    Endocrine Unit, Department of Medical Sciences, University of Milan, Fondazione Ospedale Maggiore IRCCS, Milan, Italy.

    The cAMP-protein kinase A (PKA) pathway is the major signal transduction pathway involved in melanocyte-stimulating hormone receptor-mediated signaling and melanin production, whereas its role in the control of melanocyte proliferation is still controversial. In this study, we evaluated the effects of selective activation of the different PKA regulatory subunits type 1A (R1A) and type 2B (R2B) on melanocyte proliferation. Immunohistochemistry demonstrated that normal melanocytes lacked R1A protein whereas this subunit was highly expressed in all human melanomas studied (N=20) and in six human melanoma cell lines. Pharmacological activation of the R2 subunits by the cAMP analogue 8-Cl-cAMP inhibited proliferation and increased caspase-3 activity by 68.77+/-10.5 and 72+/-9% respectively, in all cell lines with the exception of the only p53-mutated one. Similar effects were obtained by activating R2 subunits with other analogues and by silencing R1A expression. The antiproliferative and proapoptotic effects of 8-Cl-cAMP were comparable to those observed with commonly used antitumoral drugs. Moreover, 8-Cl-cAMP potentiated the effects of these drugs on both cell proliferation and caspase-3 activity. In conclusion, this study first reports that human melanomas are characterized by a high R1/R2 ratio and that pharmacological and genetic manipulations able to revert this unbalanced expression cause significant antiproliferative and proapoptotic effects in melanoma cells.

    Oncogene 2008;27;13;1834-43

  • An immortalized human cell line bearing a PRKAR1A-inactivating mutation: effects of overexpression of the wild-type Allele and other protein kinase A subunits.

    Nesterova M, Bossis I, Wen F, Horvath A, Matyakhina L and Stratakis CA

    Section on Endocrinology and Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. nesterom@mail.nih.gov

    Context: Inactivating mutations of PRKAR1A, the regulatory subunit type 1A (RIalpha) of protein kinase A (PKA), are associated with tumor formation.

    Objective: Our objective was to evaluate the role of PKA isozymes on proliferation and cell cycle.

    Methods: A cell line with RIalpha haploinsufficiency due to an inactivating PRKAR1A mutation (IVS2+1 G-->A) was transfected with constructs encoding PKA subunits. Genetics, PKA subunit mRNA and protein expression and proliferation, aneuploidy, and cell cycle status were assessed. To identify factors that mediate PKA-associated cell cycle changes, we studied E2F and cyclins expression in transfected cells and E2F's role by small interfering RNA; we also assessed cAMP levels and baseline and stimulated cAMP signaling in transfected cells.

    Results: Introduction of PKA subunits led to changes in proliferation and cell cycle: a decrease in aneuploidy and G(2)/M for the PRKAR1A-transfected cells and an increase in S phase and aneuploidy for cells transfected with PRKAR2B, a known PRKAR1A mutant (RIalphaP), and the PKA catalytic subunit. There were alterations in cAMP levels, PKA subunit expression, cyclins, and E2F factors; E2F1 was shown to possibly mediate PKA effects on cell cycle by small interfering RNA studies. cAMP levels and constitutive and stimulated cAMP signaling were altered in transfected cells.

    Conclusion: This is the first immortalized cell line with a naturally occurring PRKAR1A-inactivating mutation that is associated in vivo with tumor formation. PKA isozyme balance is critical for the control of cAMP signaling and related cell cycle and proliferation changes. Finally, E2F1 may be a factor that mediates dysregulated PKA's effects on the cell cycle.

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

    The Journal of clinical endocrinology and metabolism 2008;93;2;565-71

  • Large deletions of the PRKAR1A gene in Carney complex.

    Horvath A, Bossis I, Giatzakis C, Levine E, Weinberg F, Meoli E, Robinson-White A, Siegel J, Soni P, Groussin L, Matyakhina L, Verma S, Remmers E, Nesterova M, Carney JA, Bertherat J and Stratakis CA

    Section on Endocrinology and Genetics and Pediatric Endocrinology Training Program, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.

    Purpose: Since the identification of PRKAR1A mutations in Carney complex, substitutions and small insertions/deletions have been found in approximately 70% of the patients. To date, no germ-line PRKAR1A deletion and/or insertion exceeded a few base pairs (up to 15). Although a few families map to chromosome 2, it is possible that current sequencing techniques do not detect larger gene changes in PRKAR1A -- mutation-negative individuals with Carney complex.

    To screen for gross alterations of the PRKAR1A gene, we applied Southern hybridization analysis on 36 unrelated Carney complex patients who did not have small intragenic mutations or large aberrations in PRKAR1A, including the probands from two kindreds mapping to chromosome 2.

    Results: We found large PRKAR1A deletions in the germ-line of two patients with Carney complex, both sporadic cases; no changes were identified in the remaining patients, including the two chromosome-2-mapping families. In the first patient, the deletion is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing Carney complex. In the second patient, the deletion led to in-frame elimination of exon 3 and the expression of a shorter protein, lacking the primary site for interaction with the catalytic protein kinase A subunit. In vitro transfection studies of the mutant PRKAR1A showed impaired ability to bind cyclic AMP and activation of the protein kinase A enzyme. The patient bearing this mutation had a more-severe-than-average Carney complex phenotype that included the relatively rare psammomatous melanotic schwannoma.

    Conclusions: Large PRKAR1A deletions may be responsible for Carney complex in patients that do not have PRKAR1A gene defects identifiable by sequencing. Preliminary data indicate that these patients may have a different phenotype especially if their defect results in an expressed, abnormal version of the PRKAR1A protein.

    Funded by: Intramural NIH HHS; NICHD NIH HHS: Z01 HD000642-04

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;2;388-95

  • Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: relationship with cell proliferation.

    Mantovani G, Lania AG, Bondioni S, Peverelli E, Pedroni C, Ferrero S, Pellegrini C, Vicentini L, Arnaldi G, Bosari S, Beck-Peccoz P and Spada A

    Department of Medical Sciences, University of Milan, Fondazione Policlinico IRCCS, Milan, Italy.

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n=16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n=5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

    Experimental cell research 2008;314;1;123-30

  • Loss of expression of protein kinase a regulatory subunit 1alpha in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions.

    Zembowicz A, Knoepp SM, Bei T, Stergiopoulos S, Eng C, Mihm MC and Stratakis CA

    Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. dr.zembowicz@DermatopathologyConsultations.com

    Pigmented epithelioid melanocytoma (PEM) is a recently described entity comprising most cases previously described as "animal-type melanoma" and epithelioid blue nevus (EBN) occurring in patients with the multiple neoplasia syndrome Carney complex (CNC). Mutations of the protein kinase A regulatory subunit type 1alpha (R1alpha) (coded by the PRKAR1A gene) are found in more than half of CNC patients. In this study, we investigated whether PEM and EBN are related at the molecular level, and whether changes in the PRKAR1A gene status and the expression of the R1alpha protein may be involved in the pathogenesis of PEM and other melanocytic lesions. Histologic analysis of hematoxylin and eosin-stained sections and immunohistochemistry (IHC) with R1alpha antibody were performed on 34 sporadic PEMs, 8 CNC-associated PEMs from patients with known PRKAR1A mutations, 297 benign and malignant melanocytic tumors (127 conventional sections of 10 compound nevi, 10 Spitz nevi, 5 deep-penetrating nevi, 5 blue nevi, 6 cellular blue nevi, 2 malignant blue nevi, 3 lentigo maligna, and 86 melanomas of various types); in addition, 170 tissue microarray sections consisting of 35 benign nevi, 60 primary melanomas, and 75 metastatic melanomas, and 5 equine dermal melanomas, were examined. Histologic diagnoses were based on preexisting pathologic reports and were confirmed for this study. DNA studies [loss of heterozygosity (LOH) for the 17q22-24 locus and the PRKAR1A gene sequencing] were performed on 60 melanomas and 7 PEMs. IHC showed that R1alpha was expressed in all but one core from tissue microarrays (169/170), and in all 127 melanocytic lesions evaluated in conventional sections. By contrast, R1alpha was not expressed in the 8 EBN from patients with CNC and PRKAR1A mutations. Expression of R1alpha was lost in 28 of 34 PEMs (82%). R1alpha was expressed in the 5 equine melanomas studied. DNA studies correlated with IHC findings: there were no PRKAR1A mutations in any of the melanomas studied and the rate of LOH for 17q22-24 was less than 7%; 5 of the 7 PEMs showed extensive 17q22-24 LOH but no PRKAR1A mutations. The results support the concept that PEM is a distinct melanocytic tumor occurring in a sporadic setting and in the context of CNC. They also suggest that PEM differs from melanomas in equine melanotic disease, further arguing that the term animal-type melanoma may be a misnomer for this group of lesions. Loss of expression of R1alpha offers a useful diagnostic test that helps to distinguish PEM from lesions that mimic it histologically.

    Funded by: Intramural NIH HHS

    The American journal of surgical pathology 2007;31;11;1764-75

  • 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

  • Alpha4 integrins are type I cAMP-dependent protein kinase-anchoring proteins.

    Lim CJ, Han J, Yousefi N, Ma Y, Amieux PS, McKnight GS, Taylor SS and Ginsberg MH

    Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA.

    A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). Inhibition of PKA-AKAP interactions modulates PKA signalling. Localized PKA activation in pseudopodia of migrating cells phosphorylates alpha4 integrins to provide spatial cues governing cell motility. Here, we report that the alpha4 cytoplasmic domain is a Type I PKA-specific AKAP that is distinct from canonical AKAPs in two ways: the alpha4 interaction requires the PKA holoenzyme, and is insensitive to amphipathic peptides that disrupt most PKA-AKAP interactions. We exploited type-specific PKA anchoring peptides to create genetically encoded baits that sequester specific PKA isoforms to the mitochondria and found that mislocalization of Type I, but not Type II, PKA disrupts alpha4 phosphorylation and markedly inhibits the velocity and directional persistence of cell migration.

    Nature cell biology 2007;9;4;415-21

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • 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

  • PRKAR1A inactivation leads to increased proliferation and decreased apoptosis in human B lymphocytes.

    Robinson-White AJ, Leitner WW, Aleem E, Kaldis P, Bossis I and Stratakis CA

    Section on Endocrinology and Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892, USA.

    The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIalpha regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIalpha levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur.

    Funded by: Intramural NIH HHS

    Cancer research 2006;66;21;10603-12

  • Depletion of type IA regulatory subunit (RIalpha) of protein kinase A (PKA) in mammalian cells and tissues activates mTOR and causes autophagic deficiency.

    Mavrakis M, Lippincott-Schwartz J, Stratakis CA and Bossis I

    Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. mavrakim@mail.nih.gov

    The human PRKAR1A gene encodes the regulatory subunit 1-alpha (RIalpha) of the cAMP-dependent protein kinase A (PKA) holoenzyme. Regulation of the catalytic activity of PKA is the only well-studied function of RIalpha. Inactivating PRKAR1A mutations cause primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC), an inherited syndrome associated with abnormal skin pigmentation and multiple neoplasias, including PPNAD. Histochemistry of tissues from CNC patients is indicative of autophagic deficiency and this led us to investigate the relationship between RIalpha and mammalian autophagy. We found that fluorescently tagged RIalpha associates with late endosomes and autophagosomes in cultured cells. The number of autophagosomes in prkar1a-/- mouse embryonic fibroblasts (MEFs) was reduced compared with wild-type MEFs. RIalpha co-immunoprecipitated with mTOR kinase, a major regulator of autophagy. Phosphorylated-mTOR levels and mTOR activity were dramatically increased in prkar1a-/- mouse cells, and in HEK 293 cells with RIalpha levels reduced by siRNA. Finally, phosphorylated-mTOR levels and mTOR activity were increased in CNC cells and in PPNAD tissues. These data suggest that RIalpha deficiency decreases autophagy by the activation of mTOR, providing a molecular basis to autophagic deficiency in PPNAD.

    Human molecular genetics 2006;15;19;2962-71

  • Haploinsufficiency at the protein kinase A RI alpha gene locus leads to fertility defects in male mice and men.

    Burton KA, McDermott DA, Wilkes D, Poulsen MN, Nolan MA, Goldstein M, Basson CT and McKnight GS

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

    Carney complex (CNC) is a familial multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and cutaneous myxomas, and endocrine tumors. CNC is inherited as an autosomal dominant trait and is transmitted with greater frequency by women vs. men. Nearly two thirds of CNC patients are heterozygous for inactivating mutations in the gene encoding the protein kinase A (PKA) type I alpha regulatory subunit (RI alpha), PRKAR1. We report here that male mice heterozygous for the Prkar1a gene have severely reduced fertility. Sperm from Prkar1a heterozygous mice are morphologically abnormal and reduced in number. Genetic rescue experiments reveal that this phenotype results from elevated PKA catalytic activity in germ cells as early as the pachytene stage of spermatogenesis. Consistent with this defect in the male mutant mice, sperm from CNC patients heterozygous for PRKAR1A mutations were also found to be morphologically aberrant and decreased in number. We conclude that unregulated PKA activity in male meiotic or postmeiotic germ cells leads to structural defects in mature sperm and results in reduced fertility in mice and humans, contributing to the strikingly reduced transmission of PRKAR1A inactivating mutations by male patients with CNC.

    Funded by: NHLBI NIH HHS: R01 HL061785, R01HL61785; NICHD NIH HHS: U54 HD012629, U54HD12629

    Molecular endocrinology (Baltimore, Md.) 2006;20;10;2504-13

  • Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor.

    Carlson CR, Lygren B, Berge T, Hoshi N, Wong W, Taskén K and Scott JD

    Biotechnology Centre of Oslo, University of Oslo, Norway.

    Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14-18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.

    Funded by: NIDDK NIH HHS: DK54441

    The Journal of biological chemistry 2006;281;30;21535-45

  • Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition.

    Dao KK, Teigen K, Kopperud R, Hodneland E, Schwede F, Christensen AE, Martinez A and Døskeland SO

    Department of Biomedicine, University of Bergen, Norway.

    The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K(d) value (2.9 microM) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K(d) values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIalpha led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.

    The Journal of biological chemistry 2006;281;30;21500-11

  • Phosphorylation of RIalpha by cyclin-dependent kinase CDK 2/cyclin E modulates the dissociation of the RIalpha-RFC40 complex.

    Gupte RS, Traganos F, Darzynkiewicz Z and Lee MY

    Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, New York 10595, USA. rakhee_gupte@nymc.edu

    We have previously demonstrated that the nuclear transport of the second subunit of the Replication Factor C complex, RFC40, by the regulatory subunit, RIalpha, of PKA is cell cycle specific and impairment in this transport results in G(1) arrest. In this study, we have investigated whether the cyclin-dependent kinases play a role in regulating the RIalpha-RFC40 complex formation. In this context, we have identified RIalpha as a novel substrate for the G(1)/S-Cyclin-dependent kinase, CDK2/Cyclin E, and found that RIalpha is specifically phosphorylated at the serine residue. Treatment of MCF7 cells with a CDK inhibitor, olomoucine, resulted in a significant accumulation in the RIalpha-RFC40 complex by 3.10 +/- 0.08 fold and a parallel decrease in the RFC40-37 complex formation by 73.73 +/- 11.81%. Furthermore, in vitro phosphorylation experiments suggest that, phosphorylation of RIalpha by CDK2/CyclinE kinase promotes the dissociation of the RIalpha-RFC40 complex and that once RIalpha is phosphorylated it cannot complex with RFC40. Inhibition of the serine-threonine phosphatase, PP1, by Calyculin A, significantly reduced the RIalpha-RFC40 complex formation, substantiating the in vitro phosphorylation data. Taken together, these findings suggest that CDK2/Cyclin E may function as downstream modulator that regulates the dissociation of the RIalpha-RFC40 complex and subsequently the association of the RFC40-RFC37 complex.

    Funded by: NCI NIH HHS: R01 CA028704, R01 CA028704-27; NIGMS NIH HHS: GM31973

    Cell cycle (Georgetown, Tex.) 2006;5;6;653-60

  • 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

  • Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC).

    Foster LJ, Rudich A, Talior I, Patel N, Huang X, Furtado LM, Bilan PJ, Mann M and Klip A

    Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.

    The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, alpha-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 +/- 0.5-fold by SILAC and 2.9 +/- 0.8-fold by immunoblotting. Insulin caused GLUT4 and alpha-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably alpha-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane.

    Journal of proteome research 2006;5;1;64-75

  • Growth hormone-secreting tumors: genetic aspects and data from animal models.

    Lytras A and Tolis G

    Division of Endocrinology and Metabolism, Hippokrateion General Hospital, Athens, Greece. alytras@otenet.gr

    Hereditary cases of growth hormone (GH)-secreting tumors have been classified into three clinical entities: the multiple endocrine neoplasia type 1 (MEN1) syndrome, the Carney complex (CNC) and the isolated familial somatotropinomas (IFS). The genomic defects associated with MEN1 are all linked to various mutations of the MEN1 gene, which is located at chromosome 11q13 and codes for menin, a nuclear protein expressed in multiple tissues. Inactivation of the MEN1 gene appears to be only rarely associated with sporadic pituitary tumor development. A CNC-associated gene, the type 1 alpha regulatory subunit (R1alpha) of cAMP-dependent protein kinase A (PRKAR1A), is located at 17q23-24. A second CNC candidate gene is located at chromosome 2p15-16, with characteristics of inheritance consistent with an oncogene; however, this gene has not been identified yet. PRKAR1A mutations are infrequently associated with sporadic GH-secreting adenomas. A candidate IFS gene is located at 11q13, in proximity to the MEN1 gene, at a locus narrowed down to a 2.21-Mb area, with approximately 50 genes, that does not appear to include the MEN1 gene. Apart from the linkage of IFS to 11q13, a possible linkage to 2p16 has also been raised, although data are still inconclusive. This manuscript reviews genetic aspects of hereditary GH-secreting tumors, data from animal models resulting from the inactivation of the MEN1 and PRKAR1A tumor suppressor genes and available in vitro data regarding possible functions of menin, the product of the MEN1 gene.

    Neuroendocrinology 2006;83;3-4;166-78

  • PRKAR1A mutations in primary pigmented nodular adrenocortical disease.

    Cazabat L, Ragazzon B, Groussin L and Bertherat J

    INSERM U567, Paris, France.

    Primary Pigmented Nodular Adrenocortical Disease (PPNAD) is a rare primary bilateral adrenal defect causing corticotropin-independent Cushing's syndrome. It occurs mainly in children and young adults. Macroscopic appearance of the adrenals is characteristic with small pigmented micronodules observed in the cortex. PPNAD is most often diagnosed in patients with Carney complex (CNC), but it can also be observed in patients without other manifestations or familial history (isolated PPNAD). The CNC is an autosomal dominant multiple neoplasia syndrome characterized by the association of myxoma, spotty skin pigmentation and endocrine overactivity. One of the putative CNC genes has been identified as the gene of the regulatory R1A subunit of protein kinase A (PRKAR1A), located at 17q22-24. Germline heterozygous inactivating mutations of PRKAR1A have been reported in about 45% of patients with CNC, and up to 80% of CNC patients with Cushing's syndrome due to PPNAD. Interestingly, such inactivating germline PRKAR1A mutations have also been found in patients with isolated PPNAD. The hot spot PRKAR1A mutation termed c.709[-7-2]del6 predisposes mostly to isolated PPNAD, and is the first clear genotype/phenotype correlation described for this gene. Somatic inactivating mutations of PRKAR1A have been observed in macronodules of PPNAD and in sporadic cortisol secreting adrenal adenomas. Isolated PPNAD is a genetic heterogenous disease, and recently inactivating mutations of the gene of the phosphodiesterase 11A4 (PDE11A4) located at 2q31-2q35 have been identified in patients without PRKAR1A mutations. Interestingly, both PRKAR1A and PDE11A gene products control the cAMP signaling pathway, which can be altered at various levels in endocrine tumors.

    Pituitary 2006;9;3;211-9

  • 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

  • PRKAR1A gene mutation in patients with cardiac myxoma.

    Mabuchi T, Shimizu M, Ino H, Yamguchi M, Terai H, Fujino N, Nagata M, Sakata K, Inoue M, Yoneda T and Mabuchi H

    Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8640, Japan. maboo398@hotmail.com

    Background: PRKAR1A gene encodes the type 1A regulatory subunit of protein kinase A. The mutation of this gene causes Carney complex which is an autosomal dominant multiple neoplasia syndrome characterized by spotty pigmentations, endocrine overactivity and cardiac myxoma. We hypothesized that cardiac myxoma may be associated with PRKAR1A gene mutation and determined whether mutation in the PRKAR1A gene is the cause of familial and sporadic cardiac myxoma.

    Methods: We studied seven patients (three males and four females) with cardiac myxoma. Two of them had familial cardiac myxoma complicated with Carney complex. The other five patients were characterized as sporadic cardiac myxomas. We analyzed the PRKAR1A gene of all patients by the polymerase chain reaction (PCR)-single-strand conformation method, followed with direct sequence analysis.

    Results: We identified a novel mutation (494delTG) in exon 4A of the PRKAR1A gene in the patients with Carney complex. A 16-year-old proband had a left atrial myxoma, pituitary adenoma and skin pigmentation. His father also had left atrial myxoma and skin pigmentation. In contrast, no mutations in the PRKAR1A gene were identified in the other five patients with sporadic cardiac myxomas.

    Conclusions: These results suggest that mutation of the PRKAR1A gene may be associated with familial cardiac myxoma in Carney complex but may not be associated with sporadic cardiac myxoma.

    International journal of cardiology 2005;102;2;273-7

  • A Japanese case of familial cardiac myxoma associated with a mutation of the PRKAR1alpha gene.

    Kojima S, Sumiyoshi M, Watanabe Y, Suwa S, Matsumoto M, Nakata Y and Daida H

    Department of Cardiology, Juntendo University Shizuoka Hospital, Nagaoka, Izunokuni-shi.

    Familial cardiac myxoma is inherited as an autosomal dominant syndrome. Here, we report a Japanese case of familial cardiac myxoma identified as a genetic abnormality. The mother experienced multiple recurrence of tumors in the left atrium and left ventricle 40 months after surgical resection of a left atrial myxoma. All recurrent tumors were successfully resected. Her daughter also had a solitary myxoma in the left atrium, but she had no recurrence after the operation. Both patients had lentigines in their face but no endocrine abnormality. Molecular genetic analysis demonstrated involvement of a mutation in the PRKAR1alpha gene.

    Internal medicine (Tokyo, Japan) 2005;44;6;607-10

  • RIalpha influences cellular proliferation in cancer cells by transporting RFC40 into the nucleus.

    Gupte RS, Pozarowski P, Grabarek J, Traganos F, Darzynkiewicz Z and Lee MY

    Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.

    The regulatory subunit (RIalpha) of cAMP-dependent Protein Kinase A (PKA) is overexpressed in a variety of tumors and carcinomas such as renal cell carcinomas, pituitary tumors of the rat, malignant osteoblasts, colon carcinomas, serous ovarian tumors and primary human breast carcinomas. However, the direct relation between overexpression of RIalpha and malignancy is still unclear. We have recently identified a novel interaction between RIalpha and RFC40, the second subunit of Replication Factor C (RFC), and have demonstrated that this interaction may be associated with cell survival. Coincidentally, RFC40 is overexpressed in gestational trophoblastic diseases such as choriocarcinomas. This study was undertaken to investigate a possible functional role for both these proteins together, in DNA replication and cellular proliferation. In the course of this study, a nonconventional nuclear localization signal was identified for RIalpha. Nuclear transport of RFC40 was found to be dependent on RIalpha, and this transport appeared to be a crucial step for cell cycle progression from G1 to S phase. Impairment in the nuclear transport of RFC40 by RIalpha arrested cells in G1 phase. These findings provide evidence for a previously unknown mechanism for the nuclear transport of RFC40 and also for a novel mechanism for cellular proliferation.

    Funded by: NCI NIH HHS: R01 CA028704, R01 CA028704-27; NIGMS NIH HHS: GM31973

    Cancer biology & therapy 2005;4;4;429-37

  • The second subunit of the replication factor C complex (RFC40) and the regulatory subunit (RIalpha) of protein kinase A form a protein complex promoting cell survival.

    Gupte RS, Weng Y, Liu L and Lee MY

    Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.

    Replication Factor C (RFC) is required for the loading of Proliferating Cell Nuclear Antigen (PCNA) onto DNA during DNA replication, repair and recombination. RFC40, the second subunit of the RFC complex, and PCNA have been shown to be overexpressed in gestational trophoblastic diseases. Using RFC40 as the bait in a yeast two-hybrid screening, we have identified a novel interaction between RFC40 and the regulatory subunit (RIalpha) of cAMP-dependent Protein kinase A (PKA). The interaction sites between these two proteins were investigated and mapped to the N-terminus of RIalpha and the C-terminus of RFC40. Moreover, it was demonstrated that the C-subunit of PKA was not associated with the RFC40-RIalpha complex. Furthermore, RFC37, the third subunit of the RFC complex, competes with RIalpha and displaces it from the RFC40-RIalpha complex. Interestingly, downregulation of endogenous RIalpha by 8-chloro cAMP, in MCF7 breast cancer cells led to reduction in the amount of RFC40-RIalpha complex, together with decrease in cell survival.

    Funded by: NIGMS NIH HHS: GM31973

    Cell cycle (Georgetown, Tex.) 2005;4;2;323-9

  • Proliferation of transformed somatotroph cells related to low or absent expression of protein kinase a regulatory subunit 1A protein.

    Lania AG, Mantovani G, Ferrero S, Pellegrini C, Bondioni S, Peverelli E, Braidotti P, Locatelli M, Zavanone ML, Ferrante E, Bosari S, Beck-Peccoz P and Spada A

    Institute of Endocrine Sciences, Ospedale Maggiore, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Milan, Italy.

    The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs.

    Cancer research 2004;64;24;9193-8

  • Minireview: PRKAR1A: normal and abnormal functions.

    Bossis I and Stratakis CA

    Section on Endocrinology and Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1862, USA.

    The type 1alpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase (PKA) (coded by the PRKAR1A gene) is the main component of type I PKA, which regulates most of the serine-threonine kinase activity catalyzed by the PKA holoenzyme in response to cAMP. Carney complex (CNC), or the complex of spotty skin pigmentation, myxomas, and endocrine overactivity, is a multiple endocrine (and not only) neoplasia syndrome that is due to PRKAR1A-inactivating mutations. The R1alpha protein and PRKAR1A mRNA have been found to be up-regulated in a series of cell lines and human and rodent neoplasms, suggesting this molecule's involvement in tumorigenesis and its potential role in cell cycle regulation, growth, and/or proliferation. Alterations in PKA activity elicit a variety of effects depending on the tissue, developmental stage, degree of differentiation, and cAMP levels. In addition, RIalpha may have functions independent of PKA. The presence of inactivating germline mutations and the loss of its wild-type allele in some CNC lesions indicate that PRKAR1A might function as a tumor suppressor gene in these tissues, but could PRKAR1A be a classic tumor suppressor gene? Probably not, and this review explains why.

    Endocrinology 2004;145;12;5452-8

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • cAMP-dependent protein kinase type I regulates ethanol-induced cAMP response element-mediated gene expression via activation of CREB-binding protein and inhibition of MAPK.

    Constantinescu A, Wu M, Asher O and Diamond I

    Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, CA 94608, USA. anconst@itsa.ucsf.edu

    We have shown that the two types of cAMP-dependent protein kinase (PKA) in NG108-15 cells differentially mediate forskolin- and ethanol-induced cAMP response element (CRE)-binding protein (CREB) phosphorylation and CRE-mediated gene transcription. Activated type II PKA is translocated into the nucleus where it phosphorylates CREB. By contrast, activated type I PKA does not translocate to the nucleus but is required for CRE-mediated gene transcription by inducing the activation of other transcription cofactors such as CREB-binding protein (CBP). We show here that CBP is required for forskolin- and ethanol-induced CRE-mediated gene expression. Forskolin- and ethanol-induced CBP phosphorylation, demonstrable at 10 min, persists up to 24 h. CBP phosphorylation requires type I PKA but not type II PKA. In NG108-15 cells, ethanol and forskolin activation of type I PKA also inhibits several components of the MAPK pathway including B-Raf kinase, ERK1/2, and p90RSK phosphorylation. As a result, unphosphorylated p90RSK no longer binds to nor inhibits CBP. Moreover, MEK inhibition by PD98059 induces a significant increase of CRE-mediated gene activation. Taken together, our findings suggest that inhibition of the MAPK pathway enhances cAMP-dependent gene activation during exposure of NG108-15 cells to ethanol. This mechanism appears to involve type I PKA-dependent phosphorylation of CBP and inhibition of MEK-dependent phosphorylation of p90RSK. Under these conditions p90RSK is no longer bound to CBP, thereby promoting CBP-dependent CREB-mediated gene expression.

    Funded by: NIAAA NIH HHS: R37 AA10030

    The Journal of biological chemistry 2004;279;41;43321-9

  • 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

  • Comparative PRKAR1A genotype-phenotype analyses in humans with Carney complex and prkar1a haploinsufficient mice.

    Veugelers M, Wilkes D, Burton K, McDermott DA, Song Y, Goldstein MM, La Perle K, Vaughan CJ, O'Hagan A, Bennett KR, Meyer BJ, Legius E, Karttunen M, Norio R, Kaariainen H, Lavyne M, Neau JP, Richter G, Kirali K, Farnsworth A, Stapleton K, Morelli P, Takanashi Y, Bamforth JS, Eitelberger F, Noszian I, Manfroi W, Powers J, Mochizuki Y, Imai T, Ko GT, Driscoll DA, Goldmuntz E, Edelberg JM, Collins A, Eccles D, Irvine AD, McKnight GS and Basson CT

    Greenberg Cardiology Division, Department of Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

    Carney complex (CNC) is a familial multiple neoplasia syndrome characterized by cardiac and extracardiac myxomas in the setting of spotty skin pigmentation and endocrinopathy. We previously identified PRKAR1A (regulatory subunit 1alpha of protein kinase A) mutations in CNC. Mutational analyses of the PRKAR1A gene in 51 unrelated CNC probands now detect mutations in 65%. All mutations, except for one unique missense mutation, lead to PRKAR1A haploinsufficiency. Therefore, we studied the consequences of prkar1a haploinsufficiency in mice. Although we did not observe cardiac myxomas or altered pigmentation in prkar1a(+/-) mice, we did observe some phenotypes similar to CNC, including altered heart rate variability. Moreover, prkar1a(+/-) mice exhibited a marked propensity for extracardiac tumorigenesis. They developed sarcomas and hepatocellular carcinomas. Sarcomas were frequently associated with myxomatous differentiation. Tumors from prkar1a(+/-) mice did not exhibit prkar1a loss of heterozygosity. Thus, we conclude that although PRKAR1A haploinsufficiency does predispose to tumorigenesis, distinct secondary genetic events are required for tumor formation.

    Funded by: NHLBI NIH HHS: P50 HL62177, R01 HL061785, R01 HL61785; NICHD NIH HHS: U54 HD012629, U54-HD-12629

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;39;14222-7

  • Functional proteomics mapping of a human signaling pathway.

    Colland F, Jacq X, Trouplin V, Mougin C, Groizeleau C, Hamburger A, Meil A, Wojcik J, Legrain P and Gauthier JM

    Hybrigenics SA, 75014 Paris, France. fcolland@hybrigenics.fr

    Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

    Genome research 2004;14;7;1324-32

  • 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

  • Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex.

    Welting TJ, van Venrooij WJ and Pruijn GJ

    Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, Nijmegen, The Netherlands.

    The eukaryotic ribonuclease for mitochondrial RNA processing (RNase MRP) is mainly located in the nucleoli and belongs to the small nucleolar ribonucleoprotein (snoRNP) particles. RNase MRP is involved in the processing of pre-rRNA and the generation of RNA primers for mitochondrial DNA replication. A closely related snoRNP, which shares protein subunits with RNase MRP and contains a structurally related RNA subunit, is the pre-tRNA processing factor RNase P. Up to now, 10 protein subunits of these complexes have been described, designated hPop1, hPop4, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp30, Rpp38 and Rpp40. To get more insight into the assembly of the human RNase MRP complex we studied protein-protein and protein-RNA interactions by means of GST pull-down experiments. A total of 19 direct protein-protein and six direct protein-RNA interactions were observed. The analysis of mutant RNase MRP RNAs showed that distinct regions are involved in the direct interaction with protein subunits. The results provide insight into the way the protein and RNA subunits assemble into a ribonucleoprotein particle. Based upon these data a new model for the architecture of the human RNase MRP complex was generated.

    Nucleic acids research 2004;32;7;2138-46

  • 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

  • Merlin links to the cAMP neuronal signaling pathway by anchoring the RIbeta subunit of protein kinase A.

    Grönholm M, Vossebein L, Carlson CR, Kuja-Panula J, Teesalu T, Alfthan K, Vaheri A, Rauvala H, Herberg FW, Taskén K and Carpén O

    Biomedicum Helsinki, Department of Pathology, Neuroscience Center, Institute of Biotechnology, University of Helsinki and Helsinki University Central Hospital, 00014 Helsinki, Finland. mikaela.gronholm@helsinki.fi

    The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIbeta. Merlin and RIbeta demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.

    The Journal of biological chemistry 2003;278;42;41167-72

  • Molecular and functional analysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q losses, somatic mutations, and protein kinase A expression and activity.

    Bertherat J, Groussin L, Sandrini F, Matyakhina L, Bei T, Stergiopoulos S, Papageorgiou T, Bourdeau I, Kirschner LS, Vincent-Dejean C, Perlemoine K, Gicquel C, Bertagna X and Stratakis CA

    Department of Endocrinology, Institut Cochin, Institut National de la Santé et de la Recherche Médicale U567, CNRS UMR8104, Université Paris V, Hôpital Cochin, Paris 75014, France.

    Germ-line protein kinase A (PKA) regulatory-subunit type-Ialpha (RIalpha; PRKAR1A)-inactivating mutations and loss-of-heterozygosity (LOH) of its 17q22-24 locus have been found in Cushing syndrome (CS) caused by primary pigmented nodular adrenocortical disease (PPNAD). We examined whether somatic 17q22-24, PRKAR1A, or PKA changes are present in 44 sporadic adrenocortical tumors (29 adenomas and 15 cancers); 26 of these tumors were responsible for CS. A probe containing the PRKAR1A gene-mapped by fluorescent in situ hybridization to 17q22-24-and corresponding microsatellite markers were used to study allelic losses; PRKAR1A was sequenced in all samples. 17q22-24 losses were seen in 23 and 53% of adenomas and cancers, respectively. In three tumors, somatic, PRKAR1A-inactivating mutations were identified: (a) a nonsense mutation in exon 6 (A751G); (b) a splicing mutation (9IVS-1G/A); and (c) a transition (1050T>C) followed by a 22-bp deletion, also in exon 9; all predicted premature RIalpha protein terminations. Quantitative message and protein studies showed RIalpha down-regulation in tumors with genetic changes; their cortisol secretion pattern was similar to that of PPNAD, and they had higher PKA activity by enzymatic studies. We conclude that somatic allelic losses of the 17q22-24 region, PRKAR1A-inactivating mutations or down-regulation, and corresponding PKA activity changes are present in at least some sporadic adrenocortical tumors, especially those with a PPNAD-like clinical presentation of CS.

    Cancer research 2003;63;17;5308-19

  • Human tumors associated with Carney complex and germline PRKAR1A mutations: a protein kinase A disease!

    Stergiopoulos SG and Stratakis CA

    Section on Endocrinology and Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Building 10, Room 10N262, 10 Center Dr. MSC1862, Bethesda, MD 20892, USA.

    Carney complex (CNC) is a multiple neoplasia syndrome that consists of endocrine (thyroid, pituitary, adrenocortical and gonadal), non-endocrine (myxomas, nevi and other cutaneous pigmented lesions), and neural (schwannomas) tumors. Primary pigmented nodular adrenocortical disease (PPNAD) is the most common endocrine manifestation of CNC and the only inherited form of Cushing syndrome known to date. In the search of genes responsible for CNC, two chromosomal loci were identified; one (17q22-24) harbored the gene encoding the type I-alpha regulatory subunit (RIalpha) of protein kinase A (PKA), PRKAR1A, a critical component of the cAMP signaling pathway. Here we review CNC and the implications of this discovery for the cAMP and/or PKA's involvement in human tumorigenesis.

    FEBS letters 2003;546;1;59-64

  • Protein kinase-A activity in PRKAR1A-mutant cells, and regulation of mitogen-activated protein kinases ERK1/2.

    Robinson-White A, Hundley TR, Shiferaw M, Bertherat J, Sandrini F and Stratakis CA

    Section on Endocrinology and Genetics, Developmental Endocrinology Branch, NICHD, Bethesda, MD 20892, USA.

    Carney complex (CNC) is caused by PRKAR1A-inactivating mutations. PRKAR1A encodes the regulatory subunit type I-alpha (RIalpha) of the cAMP-dependent kinase (PKA) holoenzyme; how RIalpha insufficiency leads to tumorigenesis remains unclear. In many cells PKA inhibits the extracellular receptor kinase (ERK1/2) cascade of the mitogen-activated protein kinase (MAPK) pathway leading to inhibition of cell proliferation. We investigated whether the PKA-mediated inhibitory effect on ERK1/2 is affected in CNC cells that carry germline PRKAR1A mutations. PKA activity both at baseline and after stimulation with cAMP was augmented in cells carrying mutations. Quantitative message analysis showed that the main PKA subunits expressed were type I (RIalpha and RIbeta) but RIalpha was decreased in mutant cells. Immunoblot assays of ERK1/2 phosphorylation by the cell- and pathway-specific stimulant lysophosphatidic acid (LPA) showed activation of this pathway in a time- and concentration-dependent manner that was prevented by a specific inhibitor. There was a greater rate of growth in mutant cells; forskolin and isoproterenol inhibited LPA-induced ERK1/2 phosphorylation in normal but not in mutant cells. Forskolin inhibited LPA-induced cell proliferation and metabolism in normal cells, but stimulated these parameters in mutant cells. These data were also replicated in a pituitary tumor cell line carrying the most common PRKAR1A mutation (c.578del TG), and an in vitro construct of mutant PRKAR1A that was recently shown to lead to augmented PKA-mediated phosphorylation. We conclude that PKA activity in CNC cells is increased and that its stimulation by forskolin or isoproterenol increases LPA-induced ERK1/2 phosphorylation, cell metabolism and proliferation. Reversal of PKA-mediated inhibition of this MAPK pathway in CNC cells may contribute to tumorigenesis in this condition.

    Human molecular genetics 2003;12;13;1475-84

  • AKAP7gamma is a nuclear RI-binding AKAP.

    Brown RL, August SL, Williams CJ and Moss SB

    Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania, Rm. 1312 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6142, USA.

    Spatial regulation of protein kinase A (PKA) is accomplished by its sequestration via A-kinase anchor proteins (AKAPs). PKA activity is critical for mammalian oocyte development, suggesting that PKA must be appropriately positioned in these large cells. A screen for AKAPs in oocytes identified AKAP7gamma, an AKAP originally found in pancreas. Yeast two-hybrid analysis and co-immunoprecipitation studies showed that AKAP7gamma bound the type I PKA regulatory subunit (RI) and that the RI-binding domain overlapped the previously identified type II PKA regulatory subunit (RII) binding domain. Overexpressed AKAP7gamma localized to the nuclei of HEK 293 cells via a nuclear localization signal. In addition, endogenous AKAP7gamma protein was found in both the nucleus and cytoplasm of oocytes. This work identifies AKAP7gamma as the first nuclear AKAP to bind RI and suggests that AKAP7gamma may be responsible for positioning PKA via RI and/or RII to regulate PKA-mediated gene transcription in both somatic cells and oocytes.

    Funded by: NCRR NIH HHS: RR-2483; NICHD NIH HHS: P01 HD06274

    Biochemical and biophysical research communications 2003;306;2;394-401

  • Impaired secretion of IL-10 by T cells from patients with common variable immunodeficiency--involvement of protein kinase A type I.

    Holm AM, Aukrust P, Aandahl EM, Müller F, Taskén K and Frøland SS

    Research Institute for Internal Medicine, National Hospital, Oslo, Norway. a.m.holm@klinmed.uio.no

    Common variable immunodeficiency (CVID) is a heterogeneous group of B cell deficiency syndromes. T cell abnormalities are present in a high proportion of patients with CVID, suggesting impaired T cell-mediated stimulation of B cells. Based on the importance of IL-10 for B cell function and the involvement of the cAMP/protein kinase A type I (PKAI) system in IL-10 synthesis, we examined IL-10 secretion in T cells from CVID patients and controls, particularly focusing on possible modulatory effects of the cAMP/PKAI system. Our main findings were: 1) anti-CD3 and anti-CD3/anti-CD28 activated T cells from CVID patients secreted less IL-10 than healthy controls. This defect was not related to varying proportions of T cell subsets (e.g., CD4(+)/CD8(+), CD45RA(+)/RO(+), or CD28(-) T cells); 2) PKAI activation through the cAMP agonist 8-CPT-cAMP markedly inhibited IL-10 secretion from T cells through CD3 and CD28 activation in both patients and controls, but the sensitivity for cAMP-dependent inhibition was increased in CVID; 3) selective PKAI inhibition by Rp-8-Br-cAMPS markedly increased IL-10 secretion in anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in both patients and controls. Even at the lowest concentrations of Rp-8-Br-cAMPS, IL-10 secretion in CVID patients reached levels comparable to those in controls. Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The involvement of the cAMP/PKAI system in this defect suggests a novel target for therapeutic immunomodulation in CVID.

    Journal of immunology (Baltimore, Md. : 1950) 2003;170;11;5772-7

  • A-kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum.

    Brown PR, Miki K, Harper DB and Eddy EM

    Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

    The fibrous sheath is a unique cytoskeletal structure located in the principal piece of the sperm flagellum and is constructed of two longitudinal columns connected by closely spaced circumferential ribs. Cyclic AMP-dependent protein kinases are secured within specific cytoplasmic domains by A-kinase anchoring proteins (AKAPs), and the most abundant protein in the fibrous sheath is AKAP4. Several other fibrous sheath proteins have been identified, but how the fibrous sheath assembles is not understood. Yeast two-hybrid assays and deletion mutagenesis were used to identify AKAP4-binding proteins and to map the binding regions on AKAP4 and on the proteins identified. We found that AKAP4 binds AKAP3 and two novel spermatogenic cell-specific proteins, Fibrous Sheath Interacting Proteins 1 and 2 (FSIP1, FSIP2). Transcription of Akap4, Akap3, and Fsip1 begins in early spermatid development, whereas transcription of Fsip2 begins in late spermatocyte development. AKAP3 is synthesized in round spermatids and incorporated into the fibrous sheath concurrently with formation of the rib precursors. However, AKAP4 is synthesized and incorporated into the nascent fibrous sheath late in spermatid development. The AKAP4 precursor is processed in the flagellum and only the mature form of AKAP4 appears to bind AKAP3. These results suggest that AKAP3 is involved in organizing the basic structure of the fibrous sheath, whereas AKAP4 has a major role in completing fibrous sheath assembly.

    Biology of reproduction 2003;68;6;2241-8

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

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

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

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

    Nature biotechnology 2003;21;5;566-9

  • Genetic testing of the family with a Carney-complex member leads to successful early removal of an asymptomatic atrial myxoma in the mother of the patient.

    Aspres N, Bleasel NR and Stapleton KM

    Department of Dermatology, Liverpool Hospital, Australia. n.aspres@bigpond.com

    Carney complex is a rare cardiocutaneous syndrome with an autosomal-dominant inheritance pattern. Apart from its cutaneous manifestations of multiple blue naevi and lentigines, it can involve multiple other organ systems, particularly the heart, where myxoma tumours commonly develop and can potentially lead to serious complications such as cerebrovascular accidents and myocardial infarction. Recently, a specific mutation in the gene encoding the R1-alpha regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase A (PRKAR1alpha) has been discovered and found to be associated with a high risk of developing cardiac myxomas. We report the case of a Carney-complex family member who displayed no observable clinical or cardiac features of the disease but who was found to be positive for the PRKAR1alpha gene mutation on genetic testing. Further evaluation of this patient subsequently led to the discovery of a 3-cm atrial myxoma that had previously been undetected on cardiac assessment. This case highlights the potential benefits of using genetic screening for this disease.

    The Australasian journal of dermatology 2003;44;2;121-2

  • 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

  • A kinase anchoring protein (AKAP) interaction and dimerization of the RIalpha and RIbeta regulatory subunits of protein kinase a in vivo by the yeast two hybrid system.

    Carlson CR, Ruppelt A and Taskén K

    Department of Medical Biochemistry, Institute for Basic Medical Sciences, University of Oslo, PO Box 1112 Blindern, N-0317 Oslo, Norway. cathrine.carlson@basalmed.uio.no

    Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta:AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored.

    Journal of molecular biology 2003;327;3;609-18

  • 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

  • Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD.

    Groussin L, Kirschner LS, Vincent-Dejean C, Perlemoine K, Jullian E, Delemer B, Zacharieva S, Pignatelli D, Carney JA, Luton JP, Bertagna X, Stratakis CA and Bertherat J

    Departments of Endocrinology, Institut Cochin, INSERM U576, CNRS UMR 8104 IFR116, René Descartes-Paris V University, France.

    We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC.

    American journal of human genetics 2002;71;6;1433-42

  • PRKAR1A, one of the Carney complex genes, and its locus (17q22-24) are rarely altered in pituitary tumours outside the Carney complex.

    Sandrini F, Kirschner LS, Bei T, Farmakidis C, Yasufuku-Takano J, Takano K, Prezant TR, Marx SJ, Farrell WE, Clayton RN, Groussin L, Bertherat J and Stratakis CA

    Journal of medical genetics 2002;39;12;e78

  • Intracellular targeting of protein kinases and phosphatases.

    Alto N, Carlisle Michel JJ, Dodge KL, Langeberg LK and Scott JD

    Howard Hughes Medical Institute, Vollum Institute, Portland, Oregon 97201-3098, USA.

    Compartmentalization of kinases and phosphatases is a key determinant in the specificity of second messenger-mediated signaling events. Localization of the cAMP-dependent protein kinase (PKA) and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). This study focused on recent advances that further our understanding of AKAPs, with particular emphasis on the bidirectional regulation of signaling events by AKAP signaling complexes and their contribution to the control of actin reorganization events.

    Funded by: NIDDK NIH HHS: DK-54441

    Diabetes 2002;51 Suppl 3;S385-8

  • Regulatory subunit type I-alpha of protein kinase A (PRKAR1A): a tumor-suppressor gene for sporadic thyroid cancer.

    Sandrini F, Matyakhina L, Sarlis NJ, Kirschner LS, Farmakidis C, Gimm O and Stratakis CA

    Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1862, USA.

    The tumor-suppressor gene encoding the cyclic AMP-dependent protein kinase A type I-alpha regulatory subunit PRKAR1A has been mapped to chromosome 17 (17q22-24) and is mutated in Carney complex, a familial neoplasia syndrome that is associated with thyroid tumors. Other genes implicated in cyclic nucleotide-dependent signaling have been investigated in thyroid tumorigenesis. We studied protein kinase A (PKA) activity in noninherited follicular thyroid adenomas and follicular, papillary, and undifferentiated (anaplastic) thyroid carcinomas. We then examined these and additional thyroid tumors for losses of the 17q22-24 PRKAR1A region, mutations of the PRKAR1A gene, and expression of its peptide product. Total PKA activity was markedly increased in carcinomas over that in adenomas, whereas the ratio of free vs. total PKA activity was decreased in cancer. Consistent with these findings, the 17q22-24 region was frequently lost in cancer but not in benign adenomas. A novel inactivating mutation of the PRKAR1A gene (leading to premature termination of the predicted protein) was found in an aggressive thyroid cancer. The tumor with PRKAR1A gene mutation, as well as the tumors with 17q allelic losses, showed decreased PRKAR1A expression by immunostaining. We conclude that PRKAR1A, the most abundant regulatory subunit of protein kinase A and a principal cyclic AMP-signaling modulator, acts as a tumor-suppressor gene in sporadic thyroid cancer. Published 2002 Wiley-Liss, Inc.

    Genes, chromosomes & cancer 2002;35;2;182-92

  • Mutations of the PRKAR1A gene in Cushing's syndrome due to sporadic primary pigmented nodular adrenocortical disease.

    Groussin L, Jullian E, Perlemoine K, Louvel A, Leheup B, Luton JP, Bertagna X and Bertherat J

    Service des Maladies Endocriniennes et Métaboliques, Centre Hospitalier Universitaire (CHU) Cochin, Paris 75014, France.

    Primary pigmented nodular adrenocortical disease (PPNAD) is a cause of ACTH-independent Cushing's syndrome. This condition can be difficult to diagnose because hypercortisolism may be periodic and adrenal imaging may not demonstrate an adrenal tumor. PPNAD can be part of the Carney complex (CNC), an autosomal dominant multiple neoplasia syndrome. Germline mutations of the regulatory subunit R1A of PKA (PRKAR1A) have been observed in about 45% of CNC kindreds. To improve our understanding of sporadic PPNAD and develop a potential diagnostic tool, we investigated the genetics of patients with sporadic and isolated PPNAD. Patients undergoing surgery for bilateral ACTH-independent Cushing's syndrome in whom pathological examination revealed PPNAD were subjected to endocrinological investigations and a systematic search for other manifestations of CNC. The PRKAR1A gene was sequenced using DNA from frozen adrenal tissues and leukocytes from three patients with sporadic isolated PPNAD and using leukocyte DNA from two additional patients. Different inactivating germline mutations of the PRKAR1A gene were found in the five patients. For three cases, study of the parents' DNA demonstrated a de novo mutation. One patient presented with an unusual 2.5-cm macronodule of the right adrenal mimicking an adrenal adenoma. A somatic 16-bp deletion of PRKAR1A gene was also found in this macronodule. Inactivating germline mutations of PRKAR1A are frequent in sporadic and isolated cases of PPNAD. The wild-type allele can be inactivated by somatic mutations, consistent with the hypothesis of the gene being a tumor suppressor gene. Thus, genetic analysis can be of help to the clinician in the diagnosis of this difficult form of adrenal Cushing's syndrome.

    The Journal of clinical endocrinology and metabolism 2002;87;9;4324-9

  • Cyclic AMP-dependent signaling aberrations in macronodular adrenal disease.

    Bourdeau I and Stratakis CA

    Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-1862, USA.

    The adrenal glands are a major source of steroid hormone biosynthesis. In normal physiology, the pituitary hormone corticotropin (ACTH) regulates the secretion of glucocorticoids via its G protein-coupled receptor (ACTHR), the product of the MC2R gene. Aldosterone is another major product of the adrenal gland; its regulation is controlled mainly by the renin-angiotensin system, although ACTH plays a role, too, especially under certain pathological conditions. The adrenal gland also secretes lesser amounts of androgens and intermediate metabolites of all these steroids. Unregulated secretion of any of these hormones can be caused by tumors, adrenocortical adenomas or carcinomas, and/or bilateral (or, rarely, unilateral) hyperplasia. Cortisol-producing hyperplasia of the adrenal glands is caused by two distinct syndromes, both of which have been directly or indirectly associated with protein kinase A signaling: (i) primary pigmented nodular adrenocortical disease (PPNAD) (a micronodular form of bilateral adrenal hyperplasia), either isolated (rarely) or in the context of Carney complex, is caused (in most cases) by mutations of the PRKAR1A gene; and (ii) ACTH-independent macronodular adrenal hyperplasia (AIMAH), or massive macronodular adrenal disease (MMAD), has been associated with aberrant (ectopic) expression, and presumably regulation, of various G protein-coupled receptors. AIMAH is a rare, sporadic condition affecting predominantly middle-aged men and women with an almost equal ratio (the latter in contrast to other forms of endogenous Cushing's syndrome). Some familial cases of AIMAH have also been described, and it appears that the pathophysiological phenomena underlying AIMAH may be present in the far more common, sporadic adrenocortical tumors and, perhaps, in the nodular growth detected in the adrenal glands of the elderly in the general population. Thus, the study of ectopic receptor expression and cAMP-dependent PKA activity in AIMAH may have wider implications for adrenal and, indeed, endocrine tumorigenesis.

    Annals of the New York Academy of Sciences 2002;968;240-55

  • Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit (PRKAR1A) in patients with the "complex of spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas" (Carney complex).

    Stratakis CA

    Unit on Genetics & Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. stratakc@cc1.nichd.nih.gov

    Carney complex (CNC) is a familial multiple neoplasia syndrome associated with abnormal skin and mucosal pigmentation. The complex has features overlapping those of McCune-Albright syndrome (MAS) and the other multiple endocrine neoplasias (MENs). CNC is inherited as an autosomal dominant trait, and the responsible genes have been mapped by linkage analysis to loci at 2p16 and 17q22-24. Because of its unusual biochemical features (e.g., paradoxical responses to various endocrine signals) and its clinical similarities to MAS, genes implicated in cyclic nucleotide-dependent signaling, including GNAS1 (which is responsible for MAS), had been considered likely candidates for causing CNC. The gene encoding the protein kinase A (PKA) type I-alpha regulatory subunit (RI alpha), PRKAR1A, had been mapped to 17q22-24; loss-of-heterozygosity (LOH) analysis using polymorphic markers from this region revealed consistent changes in tumors from patients with CNC, including those from one family previously mapped to 17q22-24. Investigation of a polymorphic site within the 5' of the PRKAR1A gene showed segregation with the disease and retention of the allele bearing the disease gene in CNC tumors. Mutations of the PRKAR1A gene were also found to have occurred de novo in sporadic cases of CNC; no mutations were found in kindreds mapping to 2p16. Thus, genetic heterogeneity in CNC was confirmed; in total, 41% of all patients with CNC had mutations in the PRKAR1A gene. All mutations were frameshifts, insertions, and deletions that led to nonsense mRNA and premature termination of the predicted peptide product. Functional studies in CNC tumors suggested that inactivating mutations of the PRKAR1A gene led to nonsense mRNA decay (the mutant peptide product was not present) and were associated with dysregulated PKA activity, increased responsiveness to cAMP, and excess of type-II PKA activity. We conclude that the PRKAR1A gene, coding for the RIalpha subunit of PKA, a critical cellular component of a number of cyclic nucleotide-dependent signaling pathways, is mutated in a subset of patients with CNC. In their tumors, there is LOH of the normal allele, suggesting that normal RI-alpha may have tumor suppression function in the tissues affected by CNC. An excess of type-II PKA activity was present in affected tissues, which may be responsible for the apparent tumorigenicity of PRKAR1A mutations in endocrine tissues.

    Annals of the New York Academy of Sciences 2002;968;3-21

  • Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions.

    Kopperud R, Christensen AE, Kjarland E, Viste K, Kleivdal H and Døskeland SO

    Department of Anatomy and Cell Biology, University of Bergen, N-5009 Bergen, Norway.

    The complex of the subunits (RIalpha, Calpha) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K(d) = 0.25 microm) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RIalpha or RIIalpha was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RIalpha or RIIalpha showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.

    The Journal of biological chemistry 2002;277;16;13443-8

  • Protein kinase A type I: a target for cancer therapy.

    Tortora G and Ciardiello F

    Clinical cancer research : an official journal of the American Association for Cancer Research 2002;8;2;303-4

  • 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

  • Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the carney complex.

    Kirschner LS, Sandrini F, Monbo J, Lin JP, Carney JA and Stratakis CA

    Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, Building 10, Room 10N262, National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, MSC1862, Bethesda, MD 20892, USA.

    Carney complex (CNC) is an autosomal dominant multiple neoplasia syndrome, which has been linked to loci on 2p16 and 17q22-24. We recently reported that PRKAR1A, which codes for the type 1A regulatory subunit of protein kinase A (PKA), is a tumor suppressor gene on chromosome 17 that is mutated in some CNC families. To evaluate the spectrum of PRKAR1A mutations, we identified its genomic structure and screened for mutations in 54 CNC kindreds (34 families and 20 patients with sporadic disease). Fourteen families were informative for linkage analysis: four of four families that mapped to 17q had PRKAR1A mutations, whereas there were no mutations found in seven families exhibiting at least one recombination with 17q. In six of the latter, CNC mapped to 2p16. PRKAR1A mutations were also found in 12 of 20 non-informative families and 7 of 20 sporadic cases. Altogether, 15 distinct PRKAR1A mutations were identified in 22 of 54 kindreds (40.7%). In 14 mutations, the sequence change was predicted to lead to a premature stop codon; one altered the initiator ATG codon. Mutant mRNAs containing a premature stop codon were unstable, as a result of nonsense-mediated mRNA decay. Accordingly, the predicted truncated PRKAR1A protein products were absent in these cells. We conclude that (i) genetic heterogeneity exists in CNC; and (ii) all of the CNC alleles on 17q are functionally null mutations of PRKAR1A. CNC is the first human disease recognized to be caused by mutations of the PKA holoenzyme, a critical component of cellular signaling.

    Human molecular genetics 2000;9;20;3037-46

  • Unconventional myosin VIIA is a novel A-kinase-anchoring protein.

    Küssel-Andermann P, El-Amraoui A, Safieddine S, Hardelin JP, Nouaille S, Camonis J and Petit C

    Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, 25 rue du Dr. Roux, Institut Pasteur, 75724 Paris cedex 15, France.

    To gain an insight into the cellular function of the unconventional myosin VIIA, we sought proteins interacting with its tail region, using the yeast two-hybrid system. Here we report on one of the five candidate interactors we identified, namely the type I alpha regulatory subunit (RI alpha) of protein kinase A. The interaction of RI alpha with myosin VIIA tail was demonstrated by coimmunoprecipitation from transfected HEK293 cells. Analysis of deleted constructs in the yeast two-hybrid system showed that the interaction of myosin VIIA with RI alpha involves the dimerization domain of RI alpha. In vitro binding assays identified the C-terminal "4.1, ezrin, radixin, moesin" (FERM)-like domain of myosin VIIA as the interacting domain. In humans and mice, mutations in the myosin VIIA gene underlie hereditary hearing loss, which may or may not be associated with visual deficiency. Immunohistofluorescence revealed that myosin VIIA and RI alpha are coexpressed in the outer hair cells of the cochlea and rod photoreceptor cells of the retina. Our results strongly suggest that myosin VIIA is a novel protein kinase A-anchoring protein that targets protein kinase A to definite subcellular sites of these sensory cells.

    The Journal of biological chemistry 2000;275;38;29654-9

  • Mutations in the protein kinase A R1alpha regulatory subunit cause familial cardiac myxomas and Carney complex.

    Casey M, Vaughan CJ, He J, Hatcher CJ, Winter JM, Weremowicz S, Montgomery K, Kucherlapati R, Morton CC and Basson CT

    Molecular Cardiology Laboratory, Cardiology Division, Department of Medicine, Weill Medical College of Cornell University, New York, New York, USA.

    Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1alpha gene encoding the R1alpha regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1alpha frameshift mutations in three unrelated families result in haploinsufficiency of R1alpha and cause Carney complex. We did not detect any truncated R1alpha protein encoded by mutant PRKAR1alpha. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1alpha deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1alpha alleles and that wild-type R1alpha protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1alpha to R2beta regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1alpha haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation.

    Funded by: NCI NIH HHS: CA78895, R01 CA078895; NHLBI NIH HHS: R01 HL-61785, R01 HL061785

    The Journal of clinical investigation 2000;106;5;R31-8

  • Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex.

    Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, Cho-Chung YS and Stratakis CA

    Unit on Genetics & Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, Maryland, USA.

    Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumours and psammomatous melanotic schwannomas. CNC is inherited as an autosomal dominant trait and the genes responsible have been mapped to 2p16 and 17q22-24 (refs 6, 7). Because of its similarities to the McCune-Albright syndrome and other features, such as paradoxical responses to endocrine signals, genes implicated in cyclic nucleotide-dependent signalling have been considered candidates for causing CNC (ref. 10). In CNC families mapping to 17q, we detected loss of heterozygosity (LOH) in the vicinity of the gene (PRKAR1A) encoding protein kinase A regulatory subunit 1-alpha (RIalpha), including a polymorphic site within its 5' region. We subsequently identified three unrelated kindreds with an identical mutation in the coding region of PRKAR1A. Analysis of additional cases revealed the same mutation in a sporadic case of CNC, and different mutations in three other families, including one with isolated inherited cardiac myxomas. Analysis of PKA activity in CNC tumours demonstrated a decreased basal activity, but an increase in cAMP-stimulated activity compared with non-CNC tumours. We conclude that germline mutations in PRKAR1A, an apparent tumour-suppressor gene, are responsible for the CNC phenotype in a subset of patients with this disease.

    Nature genetics 2000;26;1;89-92

  • Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.

    Simpson JC, Wellenreuther R, Poustka A, Pepperkok R and Wiemann S

    Department of Cell Biology and Biophysics, EMBL Heidelberg, Germany.

    As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For > 80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.

    EMBO reports 2000;1;3;287-92

  • Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis.

    Reinton N, Collas P, Haugen TB, Skâlhegg BS, Hansson V, Jahnsen T and Taskén K

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

    Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s).

    Developmental biology 2000;223;1;194-204

  • 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

  • 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

  • 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

  • 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

  • Dissecting the cooperative reassociation of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Role of Trp-196 in the catalytic subunit.

    Gibson RM and Taylor SS

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

    The catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active.

    Funded by: NCI NIH HHS: T32 CA0952223-08; NIGMS NIH HHS: GM34921

    The Journal of biological chemistry 1997;272;51;31998-2005

  • 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

  • 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

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823, U54 HG003273

    Genome research 1997;7;4;353-8

  • 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

  • The RIalpha subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-receptor.

    Tortora G, Damiano V, Bianco C, Baldassarre G, Bianco AR, Lanfrancone L, Pelicci PG and Ciardiello F

    Cattedra di Oncologia Medica, Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica, Università di Napoli Federico II, Napoli, Italy.

    Functional interactions between protein kinase A (PKA) and epidermal growth factor receptor (EGF-R) signalling pathways have been suggested. Unlike the type II isoform of PKA (PKAII), the type I (PKAI) and/or its regulatory subunit RIalpha are generally overexpressed in cancer cells and are induced following transforming growth factor alpha (TGF alpha)/EGF-R-dependent transformation. Downregulation of RIalpha/PKAI inhibits TGF alpha expression and EGF-R-dependent signalling. We have previously shown that addition of EGF to quiescent human normal epithelial MCF-10A cells determines PKAI expression and cell membrane translocation before cells enter S phase, while PKAI inhibition prevents S phase entry. Constitutive overexpression of PKAI confers the ability to grow in serum free medium, bypassing EGF requirement. Here we demonstrate a direct interaction of PKAI, but not of PKAII, with the activated EGF-R, that occurs within 5 min following EGF treatment of MCF-10A cells. Moreover, induction of mitogen-activated protein kinase (MAPK) activity following EGF-R activation is mimicked by PKAI overexpression and inhibited by downregulators of PKAI. Finally, the PKAI-EGF-R association occurs through the binding of RIalpha to the SH3 domain(s) of Grb2 adaptor protein, thus allowing the recruitment of the PKAI holoenzyme to the activated EGF-R. This is the first demonstration of a direct interaction of PKAI with the activated EGF-R macromolecular signalling complex.

    Oncogene 1997;14;8;923-8

  • The human gene for the regulatory subunit RI alpha of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase: two distinct promoters provide differential regulation of alternately spliced messenger ribonucleic acids.

    Solberg R, Sandberg M, Natarajan V, Torjesen PA, Hansson V, Jahnsen T and Taskén K

    Institute of Medical Biochemistry, University of Oslo, Norway. rigmor.solberg@salmed.uio.no

    The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.

    Endocrinology 1997;138;1;169-81

  • Sporadic cardiac myxomas and tumors from patients with Carney complex are not associated with activating mutations of the Gs alpha gene.

    DeMarco L, Stratakis CA, Boson WL, Jakbovitz O, Carson E, Andrade LM, Amaral VF, Rocha JL, Choursos GP, Nordenskjöld M and Friedman E

    Department of Pharmacology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

    Cardiac myxomas are rare tumors that may be encountered sporadically or in the context of the Carney complex. The molecular basis for the development of cardiac myxomas and Carney complex tumors is unclear. Pathological myocardial function and myocardial hypertrophy have been associated with alterations in the heterotrimeric GTP-binding proteins. The postulated proto-oncogenic character of the gene encoding the alpha sub-unit of the stimulatory GTP-binding protein Gs alpha (gsp) in pituitary and thyroid tumors, the finding of identical somatic gsp mutations in the myocardium of patients with McCune-Albright syndrome, and the associated endocrine anomalies of the Carney complex prompted us to investigate the occurrence of activating missense mutations in the Gs alpha gene in 10 sporadically occurring atrial myxomas and in 8 tumors from 7 patients with Carney complex. No gsp mutations could be demonstrated by using the polymerase chain reaction and denaturing gradient gel electrophoresis complemented by direct DNA sequencing. Thus, activating Gs alpha mutations neither are associated with the development of atrial myxomas, nor can be demonstrated in other tumors from patients with Carney complex. The significance of these mutations in the myocardium of asymptomatic patients with McCune-Albright syndrome remains to be determined.

    Human genetics 1996;98;2;185-8

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Carney complex, a familial multiple neoplasia and lentiginosis syndrome. Analysis of 11 kindreds and linkage to the short arm of chromosome 2.

    Stratakis CA, Carney JA, Lin JP, Papanicolaou DA, Karl M, Kastner DL, Pras E and Chrousos GP

    Section on Pediatric Endocrinology, National Institute of Child Health & Human Development, National Institutes of Health (NIH), Bethesda, Maryland 20892, USA. stratakc@cc1.nichd.nih.gov

    Carney complex is an autosomal dominant syndrome characterized by multiple neoplasias, including myxomas at various sites and endocrine tumors, and lentiginosis. The genetic defect(s) responsible for the complex remain(s) unknown. We studied 101 subjects, including 51 affected members, from 11 North American kindreds with Carney complex. Blood samples were collected from patients and their family members. Hospital records, photographs, and tissue specimens of deceased individuals were reviewed. DNA was extracted from blood samples, patient-derived cell lines, and/or paraffin-embedded tissues. Linkage analysis was performed with highly polymorphic microsatellite markers, distributed over areas of the human genome harboring the most likely candidate genes. The most prevalent clinical manifestation in patients with Carney complex was spotty skin pigmentation, similar to that observed in Peutz-Jeghers and other lentiginosis syndromes. Skin and cardiac myxomas, Cushing syndrome, and acromegaly were present in 62, 30, 31 and 8 percent of the patients, respectively. Linkage was obtained for three markers on the short arm of chromosome 2 (2p16), with a maximum two-point lod score of 5.97 at theta = 0.03 for the marker CA-2 (odds in favor of linkage 10(6):1. The flanking markers CA7 and D2S378 defined a region of approximately 6.4 cM that is likely to contain the gene(s) associated with Carney complex. Candidate genes in the proximity, including the propiomelanocortin and the DNA-mismatch repair hMSH2 genes, were excluded. We conclude that the genetic defect(s) responsible for Carney complex map(s) to the short arm of chromosome 2 (2p16). This region has exhibited cytogenetic aberrations in atrial myxomas associated with the complex, and has been characterized by microsatellite instability in human neoplasias.

    The Journal of clinical investigation 1996;97;3;699-705

  • 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

  • Novel isozymes of cAMP-dependent protein kinase exist in human cells due to formation of RI alpha-RI beta heterodimeric complexes.

    Taskén K, Skålhegg BS, Solberg R, Andersson KB, Taylor SS, Lea T, Blomhoff HK, Jahnsen T and Hansson V

    Institute of Medical Biochemistry, University of Oslo, Norway.

    We report that a human neoplastic B cell line (Reh) contains cAMP-dependent protein kinase (cAK) type I (cAKI), but is practically devoid of cAK type II (cAKII). However, these cells contain a novel cAKI isozyme consisting of an RI alpha-RI beta heterodimer in association with phosphotransferase activity (RI alpha RI beta C2) eluting from DEAE-cellulose columns at a salt concentration characteristic of a cAKII. Immunoprecipitation of 8-azido-[32P]cAMP-labeled extracts and DEAE fractions employing specific antibodies directed against RI alpha and RI beta clearly demonstrated the presence of RI alpha-RI beta heterodimers. RI alpha was precipitated with RI beta antiserum and vice versa. Furthermore, disruption of disulfide bridges by reduction-alkylation abolished this coimmunoprecipitation. In addition, formation of heterodimeric complexes of RI alpha and RI beta could be demonstrated in vitro using recombinant RI proteins. Finally, the presence of low levels of RI alpha-RI beta heterodimers could also be demonstrated in human peripheral blood T lymphocytes. RI alpha-RI beta heterodimers complexed with the catalytic subunit represent a novel isozyme of cAKI (RI alpha RI beta C2), which enhances the possibilities for diversification of cAMP-mediated effects.

    Funded by: NIGMS NIH HHS: GM34921

    The Journal of biological chemistry 1993;268;28;21276-83

  • 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

  • Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of ret tyrosine kinase and the regulatory subunit RI alpha of cyclic AMP-dependent protein kinase A.

    Bongarzone I, Monzini N, Borrello MG, Carcano C, Ferraresi G, Arighi E, Mondellini P, Della Porta G and Pierotti MA

    Division of Experimental Oncology A, Istituto Nazionale Tumori, Milan, Italy.

    The ret oncogene frequently has been found activated in papillary thyroid carcinomas. A previous characterization of ret activation revealed recombination of its tyrosine kinase domain and sequences derived from an uncharacterized locus (D10S170). The mechanism leading to this recombination was identified as a paracentric inversion of the long arm of chromosome 10, inv(10)(q11.2q21), with the breakpoints occurring where ret and D10S170 were mapped. To further characterize the activation of ret in papillary thyroid carcinomas, we have now isolated and sequenced a second type of ret oncogenic rearrangement not involving the D10S170 locus. The nucleotide sequence indicated that the transforming activity was created by the fusion of the ret tyrosine kinase domain with part of the RI alpha regulatory subunit of protein kinase A (PKA). This is the first example of an oncogenic activity involving a PKA gene. PKA is the main intracellular cyclic AMP receptor, and its RI alpha subunit gene is located on chromosome 17q. RI alpha-ret transcripts encode two isoforms of the chimeric protein (p76 and p81), which display constitutive tyrosine phosphorylation as well as a tyrosine kinase enzymatic activity. Under nonreducing conditions, both isoforms are found in a dimeric configuration because of both homo- and heterodimer formation. Thus, the in vivo activation of ret in human papillary thyroid carcinomas is provided by the fusion of its tyrosine kinase domain with different genes and can be mediated by different mechanisms of gene rearrangement.

    Molecular and cellular biology 1993;13;1;358-66

  • The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase.

    Boshart M, Weih F, Nichols M and Schütz G

    Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.

    The tissue-specific extinguisher locus TSE1, a dominant negative regulator of transcription in somatic cell hybrids, acts via a cAMP response element (CRE) to repress activity of a hepatocyte-specific enhancer. Guided by the antagonism between TSE1 and cAMP-mediated signal transduction, we identified the regulatory subunit RI alpha of protein kinase A (PKA) as the product of the TSE1 locus. The evidence derives from concordant expression of RI alpha mRNA and TSE1 genetic activity, high resolution mapping of the RI alpha gene and TSE1 on human chromosome 17, and the ability of a transfected RI alpha cDNA to generate a phenocopy of TSE1-mediated extinction. The mechanism of TSE1/RI alpha-mediated extinction involves repression of basal PKA activity, reduced phosphorylation of CREB at Ser-133, and a corresponding reduction of in vivo protein binding at the target CRE.

    Cell 1991;66;5;849-59

  • Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A.

    Jones KW, Shapero MH, Chevrette M and Fournier RE

    Department of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.

    Tissue-specific extinguisher 1 (TSE1) is a trans-acting locus on human chromosome 17 that down-regulates expression of seven liver genes in hepatoma x fibroblast hybrids. To study the mechanism by which TSE1 functions, we used subtractive cDNA hybridization to clone transcripts encoded within a 2-4 Mb segment of chromosome 17 that includes TSE1. High resolution mapping within this region indicated that 8 of 9 different human cDNAs so obtained were distinct from TSE1. The remaining cDNA clone mapped concordantly with TSE1 in a panel of fragment-containing hybrids. DNA sequencing indicated that this cDNA encoded regulatory subunit RI alpha of cAMP-dependent protein kinase, and RI alpha mRNA levels correlated with TSE1 activity in various hybrid lines. Stable transfection of wild-type or cAMP-binding mutant RI alpha alleles into hepatoma recipients produced an extinction phenotype indistinguishable from that encoded by human TSE1. We conclude that TSE1 encodes a regulatory subunit of protein kinase A whose activity differs in different cell types.

    Funded by: NIGMS NIH HHS: GM26449

    Cell 1991;66;5;861-72

  • 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

  • The two mRNA forms for the type I alpha regulatory subunit of cAMP-dependent protein kinase from human testis are due to the use of different polyadenylation site signals.

    Sandberg M, Skålhegg B and Jahnsen T

    Laboratory for Gene Technology, University of Oslo, Norway.

    Using a cDNA probe for the type I alpha regulatory subunit, two mRNA species (1.5 and 3.0 kb in length) were detected in human testis. From a human testis cDNA-library a 3.0 kb clone, containing the entire reading frame of the protein, was isolated. Comparison of the nucleotide sequence of this clone to the sequence of a 1.5 kb cDNA clone earlier reported, showed that the longer clone was identical to the shorter but extended another 1.5 kb in the 3' end. Sequencing data together with Northern blot analysis indicated that the two mRNA species for human type I alpha regulatory subunit were generated from the same gene by the use of different polyadenylation site signals.

    Biochemical and biophysical research communications 1990;167;1;323-30

  • 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

  • Coordinate regulation of two genes encoding gluconeogenic enzymes by the trans-dominant locus Tse-1.

    Lem J, Chin AC, Thayer MJ, Leach RJ and Fournier RE

    Department of Microbiology, University of Southern California School of Medicine, Los Angeles 90033.

    Tissue-specific extinguisher-1 (Tse-1) is a mouse genetic locus that can repress liver-specific tyrosine aminotransferase gene expression in trans. To search for other Tse-1-responsive genes, hepatoma microcell hybrids retaining mouse chromosome 11 or human chromosome 17, containing murine Tse-1 and human TSE1, respectively, were screened for expression of liver-specific mRNAs. While most liver gene activity was unaffected in such hybrids, phosphoenolpyruvate carboxykinase and tyrosine aminotransferase gene expression was coordinately repressed in these clones. Extinction of both genes was apparently mediated by a single genetic locus that resides on human chromosome 17.

    Funded by: NIGMS NIH HHS: GM26449

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;19;7302-6

  • Molecular cloning, cDNA structure and deduced amino acid sequence for a type I regulatory subunit of cAMP-dependent protein kinase from human testis.

    Sandberg M, Taskén K, Oyen O, Hansson V and Jahnsen T

    Institute of Pathology, Rikshospitalet, Oslo, Norway.

    A 1.5 kilobase (kb) cDNA clone containing the entire coding region for a regulatory subunit of type I cAMP-dependent protein kinase (RI) was isolated from a human testis cDNA library. The cDNA clone encodes a protein of 381 amino acids that shows 98% and 97% homology to the bovine skeletal muscle RI and rat brain RI, respectively. Northern blot analysis demonstrates two major mRNA-species (1.5 and 3.0 kb) in human testis and one mRNA-species (3.0 kb) in human T-lymphocytes.

    Biochemical and biophysical research communications 1987;149;3;939-45

  • Exclusion of catalytic and regulatory subunits of cAMP-dependent protein kinase as candidate genes for the defect causing cystic fibrosis.

    Scambler P, Oyen O, Wainwright B, Farrall M, Law HY, Estivill X, Sandberg M, Williamson R and Jahnsen T

    Department of Biochemistry, St. Mary's Hospital Medical School, University of London, England.

    Cystic fibrosis (CF) is a common autosomal recessive disease with significant morbidity and mortality. Defects in cAMP control mechanisms are implicated in the pathophysiology of the disease. The mutation causing CF has been localized to chromosome 7q22-7q31.1. We have used (1) somatic-cell hybrids containing this region of the human genome in a mouse background and (2) segregation analysis in families to exclude both the genes coding for a catalytic subunit and three distinct regulatory subunits of cAMP-dependent protein kinase as candidates for the gene defect in CF. Two of these genes--those for the human homologue of the mouse type I regulatory subunit and the human homologue of the rat type II regulatory subunit--map to human chromosome 7.

    American journal of human genetics 1987;41;5;925-32

  • HLA-A2 antigen phosphorylation in vitro by cyclic AMP-dependent protein kinase. Sites of phosphorylation and segmentation in class i major histocompatibility complex gene structure.

    Guild BC and Strominger JL

    The heavy chain of the HLA-A2 antigen is phosphorylated by cyclic AMP-dependent protein kinase at two serine residues of the intracellular region. Limited proteolysis was performed on purified [32P]HLA-A2 antigens in order to define the sites of phosphorylation. Both of the phosphorylated serine residues are located in the carboxyl terminus of the heavy chain; one is encoded by exon 5, while the other is encoded by exon 6. The phosphoserine encoded by exon 5 is part of the conserved sequence Arg-Arg-Lys-Ser-Ser. This protein sequence contains the proper arrangement of amino acids for recognition and phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. In the murine class I antigens (H-2), exon 5 encodes a similar sequence of basic residues followed by one intervening residue and a threonine rather than a serine residue in the last amino acid position. A composite figure is presented that compares the carboxyl-terminal sequences of human and murine class I antigens and illustrates the known sites of phosphorylation recognized by various kinases. Each site of phosphorylation in the carboxyl terminus is contained within a conserved protein sequence encoded by one of the three exons. A separation and preservation of unique sites of phosphorylation could explain why there is segmentation in the genomic arrangement of class I molecules.

    Funded by: NIADDK NIH HHS: AM-13230; NIAID NIH HHS: AI-10736

    The Journal of biological chemistry 1984;259;21;13504-10

  • Phosphorylation of cAMP-dependent protein kinase subunits.

    Geahlen RL, Carmichael DF, Hashimoto E and Krebs EG

    The cAMP-dependent protein kinases comprise two enzyme forms designated as type I and type II. The type II enzyme can catalyze an autophosphorylation reaction whereby phosphate is transferred from ATP to one seryl residue on each regulatory subunit monomer. Since this reaction can occur in the absence of cAMP-induced enzyme dissociation, it has been used as a probe to identify one site of interaction between the catalytic subunit (C) and the type II regulatory subunit (R11). The type I cAMP-dependent protein kinase does not catalyze an analogous reaction; however, if cGMP-dependent protein kinase is substituted for C, the type I regulatory subunit (R1) becomes phosphorylated. The effects of cyclic nucleotides on this reaction, coupled with the ability of R1 to serve as an inhibitor of cGMP-dependent protein kinase suggest that this phosphorylation also occurs within an important functional domain on R1. A comparison of the autophosphorylation site on R11 with the cGMP-dependent protein kinase catalyzed phosphorylation site on R1 indicates that each modification takes place within a similar proteolytically sensitive region. On each subunit, this sensitive "hinge" region lies distal to the functional domain responsible for regulatory subunit dimerization and proximal to that responsible for cAMP binding. Phosphorylation of the "hinge" region decreases the affinity of each regulatory subunit for C, although the magnitude of this change appears greater for R1 than for R11. Phosphorylation of R1 also reduces the stoichiometry of cAMP binding from two to one mole of cAMP bound per mole of R1 monomer. These results suggest that the "hinge" regions of both R1 and R11 form part of the interaction site between the regulatory subunit and C; and, in the case of R1, it also forms a portion of one of two cAMP-binding sites. The amino acid sequence surrounding the phosphorylated serine of each regulatory subunit has been determined: R11: D-R-R-V-S(P)-V R1: R-R-R-R-G-A-I-S(P)-A It is thought that the number and position of the basic amino acid residues proximal to the modified serine may be responsible, in part, for determining the susceptibility of each site to phosphorylation by cAMP or cGMP-dependent protein kinase. Both R1 and R11 exist as phosphoproteins in vivo. Dephosphorylation of purified "native" phospho-R1 is without effect on the ability of R1 to interact with either C or cAMP. The site phosphorylated in vivo is therefore distinct from that modified in vitro by cGMP-dependent protein kinase. In addition to the autophosphorylation site, R11 possesses a second, less enzymatically reactive, phosphorylation site that is modified in vivo. Dephosphorylation of this site is also without apparent effect on the functional properties of R11. The kinases responsible for catalyzing the phosphorylation of R1 and the cryptic site on R11 and the role that these modifications play in modulating kinase activity are currently unknown but are under active investigation.

    Funded by: NIGMS NIH HHS: GM 07116, GM 07962

    Advances in enzyme regulation 1982;20;195-209

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
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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