G2Cdb::Gene report

Gene id
G00001443
Gene symbol
PPP3CA (HGNC)
Species
Homo sapiens
Description
protein phosphatase 3, catalytic subunit, alpha isozyme
Orthologue
G00000194 (Mus musculus)

Databases (7)

Gene
ENSG00000138814 (Ensembl human gene)
5530 (Entrez Gene)
43 (G2Cdb plasticity & disease)
PPP3CA (GeneCards)
Literature
114105 (OMIM)
Marker Symbol
HGNC:9314 (HGNC)
Protein Sequence
Q08209 (UniProt)

Synonyms (2)

  • CNA1
  • PPP2B

Literature (62)

Pubmed - other

  • Secreted frizzle-related protein 2 stimulates angiogenesis via a calcineurin/NFAT signaling pathway.

    Courtwright A, Siamakpour-Reihani S, Arbiser JL, Banet N, Hilliard E, Fried L, Livasy C, Ketelsen D, Nepal DB, Perou CM, Patterson C and Klauber-Demore N

    Departments of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    Secreted frizzle-related protein 2 (SFRP2), a modulator of Wnt signaling, has recently been found to be overexpressed in the vasculature of 85% of human breast tumors; however, its role in angiogenesis is unknown. We found that SFRP2 induced angiogenesis in the mouse Matrigel plug assay and the chick chorioallantoic membrane assay. SFRP2 inhibited hypoxia induced endothelial cell apoptosis, increased endothelial cell migration, and induced endothelial tube formation. The canonical Wnt pathway was not affected by SFRP2 in endothelial cells; however, a component of the noncanonical Wnt/Ca2+ pathway was affected by SFRP2 as shown by an increase in NFATc3 in the nuclear fraction of SFRP2-treated endothelial cells. Tacrolimus, a calcineurin inhibitor that inhibits dephosphorylation of NFAT, inhibited SFRP2-induced endothelial tube formation. Tacrolimus 3 mg/kg/d inhibited the growth of SVR angiosarcoma xenografts in mice by 46% (P = 0.04). In conclusion, SFRP2 is a novel stimulator of angiogenesis that stimulates angiogenesis via a calcineurin/NFAT pathway and may be a favorable target for the inhibition of angiogenesis in solid tumors.

    Funded by: NCI NIH HHS: 1 K08CA098034-01A2, K08 CA098034, K08 CA098034-05, P50 CA058223, P50 CA058223-160023, P50 CA058223-160024, P50-CA58223, R01 CA142657; NIAMS NIH HHS: R01 AR02030, R01 AR047901, R01 AR047901-06A2

    Cancer research 2009;69;11;4621-8

  • Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.

    Sandebring A, Thomas KJ, Beilina A, van der Brug M, Cleland MM, Ahmad R, Miller DW, Zambrano I, Cowburn RF, Behbahani H, Cedazo-Mínguez A and Cookson MR

    Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America.

    PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

    Funded by: Intramural NIH HHS

    PloS one 2009;4;5;e5701

  • A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

    Rodríguez A, Roy J, Martínez-Martínez S, López-Maderuelo MD, Niño-Moreno P, Ortí L, Pantoja-Uceda D, Pineda-Lucena A, Cyert MS and Redondo JM

    Department of Vascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain. a.rodriguez@uam.es

    The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

    Funded by: NIGMS NIH HHS: GM-48728, R01 GM048729, R01 GM048729-13, R01 GM048729-14, R01 GM048729-15, R01 GM048729-16, R01 GM048729-17, R01 GM048729-18

    Molecular cell 2009;33;5;616-26

  • The proline-rich N-terminal sequence of calcineurin Abeta determines substrate binding.

    Kilka S, Erdmann F, Migdoll A, Fischer G and Weiwad M

    Max Planck Research 36a Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany.

    Three different genes of catalytic subunit A of the Ca(2+)-dependent serine/threonine protein phosphatase calcineurin (CaN) are encoded in the human genome forming heterodimers with regulatory subunit B. Even though physiological roles of CaN have been investigated extensively, less is known about the specific functions of the different catalytic isoforms. In this study, all human CaN holoenzymes containing either the alpha, beta, or gamma isoform of the catalytic subunit (CaN alpha, beta, or gamma, respectively) were expressed for the first time. Comparative kinetic analysis of the dephosphorylation of five specific CaN substrates provided evidence that the distinct isoforms of the catalytic subunit confer substrate specificities to the holoenzymes. CaN alpha dephosphorylates the transcription factor Elk-1 with 7- and 2-fold higher catalytic efficiencies than the beta and gamma isoforms, respectively. CaN gamma exhibits the highest k(cat)/K(m) value for DARPP-32, whereas the catalytic efficiencies for the dephosphorylation of NFAT and RII peptide were 3- and 5-fold lower, respectively, when compared with the other isoforms. Elk-1 and NFAT reporter gene activity measurements revealed even more pronounced substrate preferences of CaNA isoforms. Moreover, kinetic analysis demonstrated that CaN beta exhibits for all tested protein substrates the lowest K(m) values. Enzymatic characterization of the CaN beta(P14G/P18G) variant as well as the N-terminal truncated form CaN beta(22-524) revealed that the proline-rich sequence of CaN beta is involved in substrate recognition. CaN beta(22-524) exhibits an at least 4-fold decreased substrate affinity and a 5-fold increased turnover number. Since this study demonstrates that all CaN isoforms display the same cytoplasmic subcellular distribution and are expressed in each tested cell line, differences in substrate specificities may determine specific physiological functions of the distinct isoforms.

    Biochemistry 2009;48;9;1900-10

  • Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis.

    Martins-de-Souza D, Gattaz WF, Schmitt A, Rewerts C, Marangoni S, Novello JC, Maccarrone G, Turck CW and Dias-Neto E

    Laboratório de Neurociências, Faculdade de Medicina da USP, Instituto de Psiquiatria, Universidade de São Paulo, Rua Dr. Ovídio Pires de Campos, No 785, s/n Consolação, São Paulo, SP, CEP 05403-010, Brazil. danms90@gmail.com

    Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease.

    Journal of neural transmission (Vienna, Austria : 1996) 2009;116;3;275-89

  • Multiple genetic variants along candidate pathways influence plasma high-density lipoprotein cholesterol concentrations.

    Lu Y, Dollé ME, Imholz S, van 't Slot R, Verschuren WM, Wijmenga C, Feskens EJ and Boer JM

    Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands. kevin.lu@wur.nl

    The known genetic variants determining plasma HDL cholesterol (HDL-C) levels explain only part of its variation. Three hundred eighty-four single nucleotide polymorphisms (SNPs) across 251 genes based on pathways potentially relevant to HDL-C metabolism were selected and genotyped in 3,575 subjects from the Doetinchem cohort, which was examined thrice over 11 years. Three hundred fifty-three SNPs in 239 genes passed the quality-control criteria. Seven SNPs [rs1800777 and rs5882 in cholesteryl ester transfer protein (CETP); rs3208305, rs328, and rs268 in LPL; rs1800588 in LIPC; rs2229741 in NRIP1] were associated with plasma HDL-C levels with false discovery rate (FDR) adjusted q values (FDR_q) < 0.05. Five other SNPs (rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, rs6060717 near SCAND1, and rs3213451 in MBTPS2 in women) were associated with plasma HDL-C levels with FDR_q between 0.05 and 0.2. Two less well replicated associations (rs3135506 in APOA5 and rs1800961 in HNF4A) known from the literature were also observed, but their significance disappeared after adjustment for multiple testing (P = 0.008, FDR_q = 0.221 for rs3135506; P = 0.018, FDR_q = 0.338 for rs1800961, respectively). In addition to replication of previous results for candidate genes (CETP, LPL, LIPC, HNF4A, and APOA5), we found interesting new candidate SNPs (rs2229741 in NRIP1, rs3213451 in MBTPS2, rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, and rs6060717 near SCAND1) for plasma HDL-C levels that should be evaluated further.

    Journal of lipid research 2008;49;12;2582-9

  • CHP2 activates the calcineurin/nuclear factor of activated T cells signaling pathway and enhances the oncogenic potential of HEK293 cells.

    Li GD, Zhang X, Li R, Wang YD, Wang YL, Han KJ, Qian XP, Yang CG, Liu P, Wei Q, Chen WF, Zhang J and Zhang Y

    Department of Immunology, Peking University Health Science Center, Beijing 100191, China.

    CHP2 (calcineurin B homologous protein 2) was initially identified as a tumor-associated antigen highly expressed in hepatocellular carcinoma. Its biological function remains largely unknown except for a potential role in transmembrane Na(+)/H(+) exchange. In the present study, we observed that ectopic expression of CHP2 promoted the proliferation of HEK293 cells, whereas knockdown of endogenous CHP2 expression in HepG2 inhibited cell proliferation. When inoculated into nude mice, CHP2 transfected HEK293 cells displayed markedly increased oncogenic potential. In analysis of the underlying molecular mechanisms, we found that like calcineurin B, CHP2 was able to bind to and stimulate the phosphatase activity of calcineurin A. In accord with this, CHP2-transfected cells showed increased nuclear presence of NFATc3 (nuclear factor of activated T cells) and enhanced NFAT activity. Finally, both accelerated cell proliferation and NFAT activation following CHP2 transfection could be suppressed by the calcineurin inhibitor cyclosporine A, suggesting an intrinsic connection between these events. Taken together, our results highlighted a potential role of CHP2 in tumorigenesis and revealed a novel function of CHP2 as an activator of the calcineurin/NFAT signaling pathway.

    The Journal of biological chemistry 2008;283;47;32660-8

  • Neither replication nor simulation supports a role for the axon guidance pathway in the genetics of Parkinson's disease.

    Li Y, Rowland C, Xiromerisiou G, Lagier RJ, Schrodi SJ, Dradiotis E, Ross D, Bui N, Catanese J, Aggelakis K, Grupe A and Hadjigeorgiou G

    Celera, Alameda, California, United States of America. yonghong.li@celera.com

    Susceptibility to sporadic Parkinson's disease (PD) is thought to be influenced by both genetic and environmental factors and their interaction with each other. Statistical models including multiple variants in axon guidance pathway genes have recently been purported to be capable of predicting PD risk, survival free of the disease and age at disease onset; however the specific models have not undergone independent validation. Here we tested the best proposed risk panel of 23 single nucleotide polymorphisms (SNPs) in two PD sample sets, with a total of 525 cases and 518 controls. By single marker analysis, only one marker was significantly associated with PD risk in one of our sample sets (rs6692804: P = 0.03). Multi-marker analysis using the reported model found a mild association in one sample set (two sided P = 0.049, odds ratio for each score change = 1.07) but no significance in the other (two sided P = 0.98, odds ratio = 1), a stark contrast to the reported strong association with PD risk (P = 4.64x10(-38), odds ratio as high as 90.8). Following a procedure similar to that used to build the reported model, simulated multi-marker models containing SNPs from randomly chosen genes in a genome wide PD dataset produced P-values that were highly significant and indistinguishable from similar models where disease status was permuted (3.13x10(-23) to 4.90x10(-64)), demonstrating the potential for overfitting in the model building process. Together, these results challenge the robustness of the reported panel of genetic markers to predict PD risk in particular and a role of the axon guidance pathway in PD genetics in general.

    PloS one 2008;3;7;e2707

  • The secondary structure of calcineurin regulatory region and conformational change induced by calcium/calmodulin binding.

    Shen X, Li H, Ou Y, Tao W, Dong A, Kong J, Ji C and Yu S

    Department of Chemistry and Institutes of Biomedical Science and Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, China.

    The protein serine/threonine phosphatase calcineurin (CN) is activated by calmodulin (CaM) in response to intracellular calcium mobilization. A widely accepted model for CN activation involves displacement of the CN autoinhibitory peptide (CN(467-486)) from the active site upon binding of CaM. However, CN activation requires calcium binding both to the low affinity sites of CNB and to CaM, and previous studies did not dissect the individual contributions of CNB and CaM to displacement of the autoinhibitory peptide from the active site. In this work we have produced separate CN fragments corresponding to the CNA regulatory region (CNRR(381-521), residues 381-521), the CNA catalytic domain truncated at residue 341, and the CNA-CNB heterodimer with CNA truncated at residue 380 immediately after the CNB binding helix. We show that the separately expressed regulatory region retains its ability to inhibit CN phosphatase activity of the truncated CN341 and CN380 and that the inhibition can be reversed by calcium/CaM binding. Tryptophan fluorescence quenching measurements further indicate that the isolated regulatory region inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The results provide new support for a model in which calcium binding to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. Moreover, the secondary structural content of the CNRR(381-521) was tentatively addressed by Fourier transform infrared spectroscopy. The results indicate that the secondary structure of CNRR(381-521) fragment is predominantly random coil, but with significant amount of beta-strand and alpha-helix structures.

    The Journal of biological chemistry 2008;283;17;11407-13

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Calcineurin activation is only one calcium-dependent step in cytotoxic T lymphocyte granule exocytosis.

    Grybko MJ, Bartnik JP, Wurth GA, Pores-Fernando AT and Zweifach A

    Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06268-3125, USA.

    We have tested the idea that calcineurin, a calcium-dependent phosphatase that is critical for activating cytokine gene expression in helper T cells, plays a role in lytic granule exocytosis in cytotoxic T lymphocytes (CTLs). We used TALL-104 human leukemic CTLs as a model. Our results confirm an earlier report (Dutz, J. P., Fruman, D. A., Burakoff, S. J., and Bierer, B. E. (1993) J. Immunol. 150, 2591-2598) that immunosuppressive drugs inhibit exocytosis in CTLs stimulated either via the T cell receptor (TCR) or via TCR-independent soluble agents. Of the two recently reported alternate targets of immunosuppressive drugs (Matsuda, S., Shibasaki, F., Takehana, K., Mori, H., Nishida, E., and Koyasu, S. (2000) EMBO Rep. 1, 428-434 and Matsuda, S., and Koyasu, S. (2000) Immunopharmacology 47, 119-125), JNK is not required for lytic granule exocytosis, but we were not able to exclude a role for P38. Exocytosis could be inhibited by expressing GFP fused to a C-terminal fragment of CAIN (cabin 1), but not by expressing VIVIT-GFP. Finally, expressing either full-length or truncated constitutively active mutant calcineurin A enhanced lytic granule exocytosis. However, the mutant calcineurin was unable to support exocytosis when cells were stimulated in the absence of Ca2+ influx. Taken together, our results support the idea that activation of calcineurin is required for lytic granule exocytosis but suggest that it is not the sole Ca2+-dependent step.

    Funded by: NIAID NIH HHS: R01AI054839

    The Journal of biological chemistry 2007;282;25;18009-17

  • Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening.

    Wu C, Ma MH, Brown KR, Geisler M, Li L, Tzeng E, Jia CY, Jurisica I and Li SS

    Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

    Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.

    Proteomics 2007;7;11;1775-85

  • Calcineurin mediates acetylcholinesterase expression during calcium ionophore A23187-induced HeLa cell apoptosis.

    Zhu H, Gao W, Jiang H, Wu J, Shi YF and Zhang XJ

    Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

    We previously reported that acetylcholinesterase plays a critical role in apoptosis and its expression is regulated by Ca(2+) mobilization. In the present study, we show that activated calpain, a cytosolic calcium-activated cysteine protease, and calcineurin, a calcium-dependent protein phosphatase, regulate acetylcholinesterase expression during A23187-induced apoptosis. The calpain inhibitor, calpeptin, and the calcineurin inhibitors, FK506 and cyclosporine A, inhibited acetylcholinesterase expression at both mRNA and protein levels and suppressed the activity of the human acetylcholinesterase promoter. In contrast, overexpression of constitutively active calcineurin significantly activated the acetylcholinesterase promoter. Furthermore, we identify a role for the transcription factor NFAT (nuclear factor of activated T cells), a calcineurin target, in regulating the acetylcholinesterase promoter during ionophore-induced apoptosis. Overexpression of human NFATc3 and NFATc4 greatly increased the acetylcholinesterase promoter activity in HeLa cells treated with A23187. Overexpression of constitutive nuclear NFATc4 activated the acetylcholinesterase promoter independent of A23187, whereas overexpression of dominant-negative NFAT blocked A23187-induced acetylcholinesterase promoter activation. These results indicate that calcineurin mediates acetylcholinesterase expression during apoptosis.

    Biochimica et biophysica acta 2007;1773;4;593-602

  • The LIFEdb database in 2006.

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

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

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

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

  • Truncation and activation of calcineurin A by calpain I in Alzheimer disease brain.

    Liu F, Grundke-Iqbal I, Iqbal K, Oda Y, Tomizawa K and Gong CX

    Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, 10314, USA. feiliu63@hotmail.com

    A disturbance of calcium homeostasis is believed to play an important role in the neurodegeneration of the brains of Alzheimer disease (AD) patients, but the molecular pathways by which it contributes to the disease are not well understood. Here we studied the activation of two major Ca(2+)-regulated brain proteins, calpain and calcineurin, in AD brain. We found that calpain I is activated, which in turn cleaves and activates calcineurin in AD brain. Mass spectrometric analysis indicated that the cleavage of calcineurin by calpain I is at lysine 501, a position C-terminal to the autoinhibitory domain, which produces a 57-kDa truncated form. The 57-kDa calcineurin maintains its Ca(2+)/calmodulin dependence of the phosphatase activity, but the phosphatase activity is remarkably activated upon truncation. The cleavage and activation of calcineurin correlate to the number of neurofibrillary tangles in human brains. These findings suggest that the overactivation of calpain I and calcineurin may mediate the role of calcium homeostatic disturbance in the neurodegeneration of AD.

    Funded by: NIA NIH HHS: AG19158

    The Journal of biological chemistry 2005;280;45;37755-62

  • 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

  • The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit.

    Buch MH, Pickard A, Rodriguez A, Gillies S, Maass AH, Emerson M, Cartwright EJ, Williams JC, Oceandy D, Redondo JM, Neyses L and Armesilla AL

    Division of Cardiology, The University of Manchester, Stopford Bldg., Manchester, UK.

    The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

    Funded by: Medical Research Council: G0200020

    The Journal of biological chemistry 2005;280;33;29479-87

  • Calcium signal-induced cofilin dephosphorylation is mediated by Slingshot via calcineurin.

    Wang Y, Shibasaki F and Mizuno K

    Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

    Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L.

    The Journal of biological chemistry 2005;280;13;12683-9

  • The linker region joining the catalytic and the regulatory domains of CnA is essential for binding to NFAT.

    Rodríguez A, Martínez-Martínez S, López-Maderuelo MD, Ortega-Pérez I and Redondo JM

    Centro Nacional de Investigaciones Cardiovasculares (CNIC), Ronda de Poniente 5, Tres Cantos, Madrid 28760, Spain.

    Calcineurin (CN) is an important regulator of developmental processes and in adults controls the immune response through its regulation of nuclear factor of activated T cells (NFAT). The physical interaction between CN and NFATs is an essential step in the activation of NFAT-dependent genes by calcium signals. Using deletional and substitutional analyses, we have identified a 13-amino acid region within CN that is essential for the interaction with NFAT and with two other CN-binding proteins, AKAP79 and Cabin-1. The interaction of CN with these proteins is selectively disrupted by substitution of specific amino acid residues within this region, indicating that NFAT and other CN-interacting proteins bind differentially to CN. This selectivity suggests that the region identified in CN could be a potential molecular target for immunosuppressive and other therapeutic interventions in diseases involving the CN/NFAT pathway.

    The Journal of biological chemistry 2005;280;11;9980-4

  • A reassessment of the inhibitory capacity of human FKBP38 on calcineurin.

    Weiwad M, Edlich F, Erdmann F, Jarczowski F, Kilka S, Dorn M, Pechstein A and Fischer G

    Max-Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany.

    The microbial peptidomacrolide FK506 affects many eukaryotic developmental and cell signaling programs via calcineurin inhibition. Prior formation of a complex between FK506 and intracellular FK506-binding proteins (FKBPs) is the precondition for the interaction with calcineurin. A puzzling difference has emerged between the mammalian multidomain protein hFKBP38 and other FKBPs. It was shown that hFKBP38 not only binds to calcineurin but also inhibits the protein phosphatase activity of calcineurin on its own [Shirane, M. and Nakayama, K.I. (2003) Nature Cell Biol. 5, 28-37]. Inherent calcineurin inhibition by hFKBP38 would completely eliminate the need for FK506 in controlling many signal transduction pathways. To address this issue, we have characterized the functional and physical interactions between calcineurin and hFKBP38. A recombinant hFKBP38 variant and endogenous hFKBP38 were tested both in vitro and in vivo. The proteins neither directly inhibited calcineurin activity nor affected NFAT reporter gene activity in SH-SY5Y and Jurkat cells. In addition, a direct physical interaction between calcineurin and hFKBP38 was not detected in co-immunoprecipitation experiments. However, hFKBP38 indirectly affected the subcellular distribution of calcineurin by interaction with typical calcineurin ligands, as exemplified by the anti-apoptotic protein Bcl-2. Our data suggest that hFKBP38 cannot substitute for the FKBP/FK506 complex in signaling pathways controlled by the protein phosphatase activity of calcineurin.

    FEBS letters 2005;579;7;1591-6

  • The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector.

    Screaton RA, Conkright MD, Katoh Y, Best JL, Canettieri G, Jeffries S, Guzman E, Niessen S, Yates JR, Takemori H, Okamoto M and Montminy M

    Peptide Biology Laboratories, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

    Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.

    Cell 2004;119;1;61-74

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

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

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

    Funded by: PHS HHS: N01-C0-12400

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

  • From ORFeome to biology: a functional genomics pipeline.

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

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

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

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

  • Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses.

    Lee SY, Wenk MR, Kim Y, Nairn AC and De Camilli P

    Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA.

    Synaptojanin 1 is a polyphosphoinositide phosphatase concentrated in presynaptic nerve terminals, where it dephosphorylates a pool of phosphatidylinositol 4,5-bisphosphate implicated in synaptic vesicle recycling. Like other proteins with a role in endocytosis, synaptojanin 1 undergoes constitutive phosphorylation in resting synapses and stimulation-dependent dephosphorylation by calcineurin. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes. Cdk5 phosphorylation inhibited the inositol 5-phosphatase activity of synaptojanin 1, whereas dephosphorylation by calcineurin stimulated such activity. The activity of synaptojanin 1 was also stimulated by its interaction with endophilin 1, its major binding partner at the synapse. Notably, Cdk5 phosphorylated serine 1144, which is adjacent to the endophilin binding site. Mutation of serine 1144 to aspartic acid to mimic phosphorylation by Cdk5 inhibited the interaction of synaptojanin 1 with endophilin 1. These results suggest that Cdk5 and calcineurin may have an antagonistic role in the regulation of synaptojanin 1 recruitment and activity, and therefore in the regulation of phosphatidylinositol 4,5-bisphosphate turnover at synapses.

    Funded by: NCI NIH HHS: CA46128, P01 CA046128; NIDA NIH HHS: DA10044, P01 DA010044; NINDS NIH HHS: NS36251, R01 NS036251, R37 NS036251

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;2;546-51

  • Phosphorylation of calcipressin 1 increases its ability to inhibit calcineurin and decreases calcipressin half-life.

    Genescà L, Aubareda A, Fuentes JJ, Estivill X, De La Luna S and Pérez-Riba M

    Genes and Disease Program, Centre de Regulació Genòmica-CRG, Passeig Marítim 37-49, 08003-Barcelona, Spain.

    Calcipressin 1 is an endogenous inhibitor of calcineurin, which is a serine/threonine phosphatase under the control of Ca(2+) and calmodulin. Calcipressin 1 is encoded by DSCR1, a gene on human chromosome 21 with seven exons, exons 1-4 are alternative first exons (isoforms 1-4). We show that calcipressin 1 isoform 1 has an N-terminal coding region longer than that previously described, and this generates a new polypeptide of 252 amino acids. This polypeptide is able to interact with calcineurin A and to inhibit NF-AT-mediated transcriptional activation. We demonstrate for the first time that endogenous calcipressin 1 exists as a complex together with the calcineurin A and B heterodimer. Calcipressin 1 is a phosphoprotein that increases its capacity to inhibit calcineurin when phosphorylated at the FLISPP motif, and this phosphorylation also controls the half-life of calcipressin 1 by accelerating its degradation. Additionally, we have also detected further phosphorylation sites outside the FLISPP motif and these contribute to the complex phosphorylation pattern of calcipressin 1. Taking all these results into consideration we suggest that phosphorylation of calcipressin 1 is involved in the regulation of the phosphatase activity of calcineurin and can therefore act as a modulator of calcineurin-dependent cellular pathways.

    The Biochemical journal 2003;374;Pt 2;567-75

  • Interaction of calcineurin and type-A GABA receptor gamma 2 subunits produces long-term depression at CA1 inhibitory synapses.

    Wang J, Liu S, Haditsch U, Tu W, Cochrane K, Ahmadian G, Tran L, Paw J, Wang Y, Mansuy I, Salter MM and Lu YM

    Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Canada, T2N 4N1.

    Long-term depression (LTD) is an activity-dependent weakening of synaptic efficacy at individual inhibitory synapses, a possible cellular model of learning and memory. Here, we show that the induction of LTD of inhibitory transmission recruits activated calcineurin (CaN) to dephosphorylate type-A GABA receptor (GABA(A)Rs) via the direct binding of CaN catalytic domain to the second intracellular domain of the GABA(A)R-gamma(2) subunits. Prevention of the CaN-GABA(A) receptor complex formation by expression of an autoinhibitory domain of CaN in the hippocampus of transgenic mice blocks the induction of LTD. Conversely, genetic expression of the CaN catalytic domain in the hippocampus depresses inhibitory synaptic responses, occluding LTD. Thus, an activity-dependent physical and functional interaction between CaN and GABA(A) receptors is both necessary and sufficient for inducing LTD at CA1 individual inhibitory synapses.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;3;826-36

  • Regulation of NF-kappaB activation by protein phosphatase 2B and NO, via protein kinase A activity, in human monocytes.

    Bengoechea-Alonso MT, Pelacho B, Osés-Prieto JA, Santiago E, López-Moratalla N and López-Zabalza MJ

    Department of Biochemistry, University of Navarra, 31080 Pamplona, Spain.

    It has been reported previously that a short synthetic immunomodulating peptide (Pa) and the neuropeptide beta-endorphin modulate the immune system. We have found now that NF-kappaB participates in the stimulation of monocytes by both peptides and we investigated the molecular mechanism by which these stimuli activate NF-kappaB. Pa and beta-endorphin induce accumulation of cyclic 3('),5(')-adenosine monophosphate (cAMP) in a calcium/calmodulin-dependent fashion since it was completely inhibited by the calmodulin antagonist W-7. The effect of these complexes seems to be mediated, at least in part, by nitric oxide (NO) synthesized by constitutive NO synthase since the NO synthase inhibitor N-methyl-L-arginine (NMLA) reduced the elevation of cAMP. Furthermore, the NO donor SIN-1 provoked nitration of G(S)alpha, leading to the cAMP elevation that was suppressed by the G(S)alpha-selective antagonist NF-449. Interestingly, the rapid degradation of NF-kappaB inhibitor IkappaBalpha induced by Pa- and beta-endorphin was reversed by a pretreatment with H-89 and cyclosporin A, inhibitors of protein kinase A (PKA) and protein phosphatase 2B (PP2B), respectively. These observations are consistent with the inhibition caused by W-7, NMLA, H-89, and cyclosporin A on NF-kappaB induction by these agonists, indicating the involvement of PKA and PP2B in the regulation of NF-kappaB in human monocytes.

    Nitric oxide : biology and chemistry 2003;8;1;65-74

  • Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis.

    Shirane M and Nakayama KI

    Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.

    The mitochondrial localization of the membrane proteins Bcl-2 and Bcl-x(L) is essential for their anti-apoptotic function. Here we show that mitochondrial FK506-binding protein 38 (FKBP38), unlike FKBP12, binds to and inhibits calcineurin in the absence of the immunosuppressant FK506, suggesting that FKBP38 is an inherent inhibitor of this phosphatase. FKBP38 is associated with Bcl-2 and Bcl-x(L) in immunoprecipitation assays and colocalizes with these proteins in mitochondria; in addition, the expression of FKBP38 mutant proteins induces a marked redistribution of Bcl-2 and Bcl-x(L). Overexpression of FKBP38 blocks apoptosis, whereas functional inhibition of this protein by a dominant-negative mutant or by RNA interference promotes apoptosis. Thus, FKBP38 might function to inhibit apoptosis by anchoring Bcl-2 and Bcl-x(L) to mitochondria.

    Nature cell biology 2003;5;1;28-37

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

    Bennasser Y, Badou A, Tkaczuk J and Bahraoui E

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

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

    Virology 2002;303;1;174-80

  • Novel human ZAKI-4 isoforms: hormonal and tissue-specific regulation and function as calcineurin inhibitors.

    Cao X, Kambe F, Miyazaki T, Sarkar D, Ohmori S and Seo H

    Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.

    We identified a thyroid hormone [3,5,3'-tri-iodothyronine (T(3))]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, alpha, beta1 and beta2, in human brain by 5'- and 3'-RACE (rapid amplification of cDNA ends). The alpha transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that beta1 and beta2 encode an identical protein product, beta, which differs from alpha in its N-terminus. Since alpha and beta contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the alpha transcript is expressed exclusively in the brain, whereas beta transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both alpha and beta transcripts, raising the question of which transcript is up-regulated by T(3). It was revealed that T(3) markedly induced the expression of alpha isoform but not of beta. This T(3)-mediated increase in the alpha isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting a complex signalling network through inhibition of calcineurin.

    The Biochemical journal 2002;367;Pt 2;459-66

  • Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes.

    Huai Q, Kim HY, Liu Y, Zhao Y, Mondragon A, Liu JO and Ke H

    Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7260, USA.

    Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the common target for two immunophilin-immunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FKBP-FK506. How the two structurally distinct immunophilin-drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-A resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike FKBP-FK506, CyPA-CsA interacts with Arg-122 at the active site of CN, implying direct involvement of CyPA-CsA in the regulation of CN catalysis. The simultaneous interaction of CyPA with both the composite surface and the active site of CN suggests that the composite surface may serve as a substrate recognition site responsible for the narrow substrate specificity of CN. The comparison of CyPA-CsA-CN with FKBP-FK506-CN significantly contributes to understanding the molecular basis of regulation of CN activity by the immunophilin-immunosuppressant.

    Funded by: NIAID NIH HHS: AI33072; NIGMS NIH HHS: GM55783

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;19;12037-42

  • Multiple domains of MCIP1 contribute to inhibition of calcineurin activity.

    Vega RB, Yang J, Rothermel BA, Bassel-Duby R and Williams RS

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. Calcineurin-dependent signals are transduced to the nucleus by nuclear factor of activated T-cells (NFAT) transcription factors that undergo nuclear translocation upon dephosphorylation and promote transcriptional activation of target genes. Several endogenous proteins are capable of inhibiting the catalytic activity of calcineurin. Modulatory calcineurin interacting protein 1 (MCIP1) is unique among these proteins on the basis of its pattern of expression and its function in a negative feedback loop to regulate calcineurin activity. Here we show that MCIP1 can be phosphorylated by MAPK and glycogen synthase kinase-3 and that phosphorylated MCIP1 is a substrate for calcineurin. Peptides corresponding to the substrate domain competitively inhibit calcineurin activity in vitro. However, a detailed structure/function analysis of MCIP1 reveals that either of two additional domains of MCIP1 is sufficient for binding to calcineurin in vitro and for inhibition of calcineurin activity in vivo. We conclude that MCIP1 inhibits calcineurin through mechanisms that include, but are not limited to, competition with other substrates such as nuclear factor of activated T-cells.

    Funded by: NHLBI NIH HHS: HL06296, HLO7360; NIAMS NIH HHS: AR40849

    The Journal of biological chemistry 2002;277;33;30401-7

  • Protein kinase Czeta phosphorylates nuclear factor of activated T cells and regulates its transactivating activity.

    San-Antonio B, Iñiguez MA and Fresno M

    Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.

    Although several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCzeta in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCzeta) or "kinase-dead" mutant (J-PKCzeta(mut)) versions of this protein. Expression of PKCzeta but not PKCzeta(mut) increased transcriptional activation mediated by the NF-kappaB or nuclear factor of activated T cells (NFAT). PKCzeta cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCzeta cells. Our results show that PKCzeta enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCzeta synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCzeta and NFAT1 or NFAT2 isoforms. Even more, PKCzeta was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1-385) protein. These data reveal a new role of PKCzeta in T cells through the control of NFAT function by modulating the activity of its transactivation domain.

    The Journal of biological chemistry 2002;277;30;27073-80

  • Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the alpha or beta catalytic subunit.

    Perrino BA, Wilson AJ, Ellison P and Clapp LH

    Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA. perrino@physio.unr.edu

    The calcineurin (CaN) alpha and beta catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the alpha or beta catalytic subunit were compared using in vitro phosphatase assays. CaN containing the alpha isoform (CnA alpha) has lower K(m) and higher V(max) values than CaN containing the beta isoform (CnA beta) toward the PO4-RII, PO4-DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the alpha or beta catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K(m) concentrations, except for a five- to ninefold higher IC50 value measured for CaN containing the beta isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the alpha isoform is more sensitive to inhibition than CaN containing the beta isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of beta CaN using PO4-DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the alpha or beta catalytic subunit isoforms suggest that the alpha:beta and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the alpha and beta catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.

    Funded by: NIDDK NIH HHS: DK-57168; NINDS NIH HHS: NS-36318

    European journal of biochemistry 2002;269;14;3540-8

  • Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins.

    Frey N and Olson EN

    Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148, USA.

    The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton. In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals. Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind alpha-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulin-dependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification. Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers. Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins alpha-actinin, gamma-filamin, and telethonin. In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc. Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.

    The Journal of biological chemistry 2002;277;16;13998-4004

  • Deletion of calcineurin and myocyte enhancer factor 2 (MEF2) binding domain of Cabin1 results in enhanced cytokine gene expression in T cells.

    Esau C, Boes M, Youn HD, Tatterson L, Liu JO and Chen J

    Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, 40 Ames St., Cambridge, MA 02139, USA.

    Cabin1 binds calcineurin and myocyte enhancer factor 2 (MEF2) through its COOH-terminal region. In cell lines, these interactions were shown to inhibit calcineurin activity after T cell receptor (TCR) signaling and transcriptional acti a1e vation of Nur77 by MEF2. The role of these interactions under physiological conditions was investigated using a mutant mouse strain that expresses a truncated Cabin1 lacking the COOH-terminal calcineurin and MEF2 binding domains. T and B cell development and thymocyte apoptosis were normal in mutant mice. In response to anti-CD3 stimulation, however, mutant T cells expressed significantly higher levels of interleukin (IL)-2, IL-4, IL-9, IL-13, and interferon gamma than wild-type T cells. The enhanced cytokine gene expression was not associated with change in nuclear factor of activated T cells (NF-AT)c or NF-ATp nuclear translocation but was preceded by the induction of a phosphorylated form of MEF2D in mutant T cells. Consistent with the enhanced cytokine expression, mutant mice had elevated levels of serum immunoglobulin (Ig)G1, IgG2b, and IgE and produced more IgG1 in response to a T cell-dependent antigen. These findings suggest that the calcineurin and MEF2 binding domain of Cabin1 is dispensable for thymocyte development and apoptosis, but is required for proper regulation of T cell cytokine expression probably through modulation of MEF2 activity.

    Funded by: NIAID NIH HHS: AI 40146, AI 44478, R01 AI040146; NIGMS NIH HHS: GM 55783

    The Journal of experimental medicine 2001;194;10;1449-59

  • Superoxide dismutase mutations of familial amyotrophic lateral sclerosis and the oxidative inactivation of calcineurin.

    Volkel H, Scholz M, Link J, Selzle M, Werner P, Tunnemann R, Jung G, Ludolph AC and Reuter A

    Department of Neurology, University of Ulm, Germany.

    Approximately 10% of all familial cases of amyotrophic lateral sclerosis (fALS) are linked to mutations in the SOD1 gene, which encodes the copper/zinc superoxide dismutase (CuZnSOD). Recently, wild-type CuZnSOD was shown to protect calcineurin, a calcium/calmodulin-regulated phosphoprotein phosphatase, from inactivation by reactive oxygen species. We asked whether the protective effect of CuZnSOD on calcineurin is affected by mutations associated with fALS. For this, we monitored calcineurin activity in the presence of mutant and wild-type SOD. We found that the degree of protection against inactivation of calcineurin by different SOD mutants correlates with the severity of the phenotype associated with the different mutations, suggesting a potential role for calcineurin-SOD1 interaction in the etiology of fALS.

    FEBS letters 2001;503;2-3;201-5

  • Caspase-mediated calcineurin activation contributes to IL-2 release during T cell activation.

    Mukerjee N, McGinnis KM, Gnegy ME and Wang KK

    Laboratory of Neuro-biochemistry, Department of CNS Molecular Sciences, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA.

    Calcineurin, a Ca(2+)/calmodulin-dependent Ser/Thr phosphatase (protein phosphatase 2B), plays a critical role in IL-2 production during T cell activation. It has been previously reported that IL-2 release in activated Jurkat T requires caspase-like activity (Posmantur et al. (1998) Exp. Cell. Res. 244, 302-309). We report here that the 60-kDa catalytic subunit of calcineurin A (Cn A) was partially cleaved to a 45-kDa form in phytohemagglutinin A (PHA) or phorbol ester + ionomycin (P + I)-activated Jurkat cells. In parallel, proteolytic activation of upstream caspases (caspase-8 and -9) as well as effector caspase-3 was also observed. Cn A cleavage was caspase mediated, since it was inhibitable by pan-caspase inhibitor Cbz-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB). Cn A cleavage was also observed when purified calcineurin was digested in vitro with caspase-3. Truncated Cn A was associated with enhanced phosphatase activity and reduced calmodulin sensitivity. Furthermore, in PHA or P + I-activated Jurkat cells, dephosphorylation of calcineurin substrate NFATc (a transcription factor known to be involved in transactivation of the IL-2 gene), was also suppressed by Z-D-DCB. Taken together, our results suggest that caspase-mediated cleavage of Cn A contributes to IL-2 production during T cell activation.

    Biochemical and biophysical research communications 2001;285;5;1192-9

  • Signals transduced by Ca(2+)/calcineurin and NFATc3/c4 pattern the developing vasculature.

    Graef IA, Chen F, Chen L, Kuo A and Crabtree GR

    Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.

    Vascular development requires an orderly exchange of signals between growing vessels and their supporting tissues, but little is known of the intracellular signaling pathways underlying this communication. We find that mice with disruptions of both NFATc4 and the related NFATc3 genes die around E11 with generalized defects in vessel assembly as well as excessive and disorganized growth of vessels into the neural tube and somites. Since calcineurin is thought to control nuclear localization of NFATc proteins, we introduced a mutation into the calcineurin B gene that prevents phosphatase activation by Ca(2+) signals. These CnB mutant mice exhibit vascular developmental abnormalities similar to the NFATc3/c4 null mice. We show that calcineurin function is transiently required between E7.5 and E8.5. Hence, early calcineurin/NFAT signaling initiates the later cross-talk between vessels and surrounding tissues that pattern the vasculature.

    Cell 2001;105;7;863-75

  • Expression of ZAKI-4 messenger ribonucleic acid in the brain during rat development and the effect of hypothyroidism.

    Siddiq A, Miyazaki T, Takagishi Y, Kanou Y, Hayasaka S, Inouye M, Seo H and Murata Y

    Department of Teratology and Genetics, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.

    We identified ZAKI-4 (also designated as DSCR1L1) as a thyroid hormone responsive gene in cultured human skin fibroblasts. Recently it has been reported that ZAKI-4 belongs to an evolutionary conserved family of proteins that function as calcineurin inhibitor. In human, ZAKI-4 and calcineurin are highly expressed in brain, where thyroid hormones play essential roles in the development during fetal and neonatal periods. In the present study, we examined the temporal and spatial expression patterns of ZAKI-4 messenger RNA (mRNA) in control and hypothyroid rat brains. Northern blot analysis revealed that ZAKI-4 mRNA was detected in both cerebral cortex and cerebellum as early as embryonic day (E)18. In the cerebral cortex, the expression level gradually increased with age, reaching a plateau at postnatal day (P)7 and remained constant thereafter until P30. A similar pattern of increase with age was also observed in hypothyroid rats; however, the magnitude of the increase was significantly reduced. In control rats, the fold increase in ZAKI-4 mRNA level from E18 to P17 was 10.8; whereas in hypothyroid rats, it was 7.4. In cerebellum the expression level did not change with age or by thyroid status. In situ hybridization revealed that ZAKI-4 mRNA is widely expressed in neurons throughout the brain. It is noteworthy that the expression in the neurons of layer VI of the cerebral cortex was more evident in control rats than that in hypothyroid rats from P17 to P30. Though not influenced by hypothyroidism, there were several regions of the brain in which ZAKI-4 mRNA was strongly expressed. These regions were the mitral cell layer of the olfactory bulb, the substantia nigra, and the hippocampus, where calcineurin is also abundantly expressed. Therefore, it may be hypothesized that ZAKI-4 plays an important role in the development and function of the brain by modulating calcineurin function; and decrease in ZAKI-4 mRNA expression in the specific brain areas may explain, in some parts, the mechanism of abnormal brain development by hypothyroidism.

    Endocrinology 2001;142;5;1752-9

  • Calsarcins, a novel family of sarcomeric calcineurin-binding proteins.

    Frey N, Richardson JA and Olson EN

    Departments of Molecular Biology and Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.

    The calcium- and calmodulin-dependent protein phosphatase calcineurin has been implicated in the transduction of signals that control the hypertrophy of cardiac muscle and slow fiber gene expression in skeletal muscle. To identify proteins that mediate the effects of calcineurin on striated muscles, we used the calcineurin catalytic subunit in a two-hybrid screen for cardiac calcineurin-interacting proteins. From this screen, we discovered a member of a novel family of calcineurin-interacting proteins, termed calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells. Calsarcin-1 and calsarcin-2 are expressed in developing cardiac and skeletal muscle during embryogenesis, but calsarcin-1 is expressed specifically in adult cardiac and slow-twitch skeletal muscle, whereas calsarcin-2 is restricted to fast skeletal muscle. Calsarcins represent a novel family of sarcomeric proteins that link calcineurin with the contractile apparatus, thereby potentially coupling muscle activity to calcineurin activation.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;26;14632-7

  • DNA cloning using in vitro site-specific recombination.

    Hartley JL, Temple GF and Brasch MA

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

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

    Genome research 2000;10;11;1788-95

  • DSCR1, overexpressed in Down syndrome, is an inhibitor of calcineurin-mediated signaling pathways.

    Fuentes JJ, Genescà L, Kingsbury TJ, Cunningham KW, Pérez-Riba M, Estivill X and de la Luna S

    Down Syndrome Research Group, Medical and Molecular Genetics Center, IRO, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain.

    Down syndrome is one of the major causes of mental retardation and congenital heart malformations. Other common clinical features of Down syndrome include gastrointestinal anomalies, immune system defects and Alzheimer's disease pathological and neurochemical changes. The most likely consequence of the presence of three copies of chromosome 21 is the overexpression of its resident genes, a fact which must underlie the pathogenesis of the abnormalities that occur in Down syndrome. Here we show that DSCR1, the product of a chromosome 21 gene highly expressed in brain, heart and skeletal muscle, is overexpressed in the brain of Down syndrome fetuses, and interacts physically and functionally with calcineurin A, the catalytic subunit of the Ca(2+)/calmodulin-dependent protein phosphatase PP2B. The DSCR1 binding region in calcineurin A is located in the linker region between the calcineurin A catalytic domain and the calcineurin B binding domain, outside of other functional domains previously defined in calcineurin A. DSCR1 belongs to a family of evolutionarily conserved proteins with three members in humans: DSCR1, ZAKI-4 and DSCR1L2. We further demonstrate that overexpression of DSCR1 and ZAKI-4 inhibits calcineurin-dependent gene transcription through the inhibition of NF-AT translocation to the nucleus. Together, these results suggest that members of this newly described family of human proteins are endogenous regulators of calcineurin-mediated signaling pathways and as such, they may be involved in many physiological processes.

    Human molecular genetics 2000;9;11;1681-90

  • Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD.

    Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF and Reed JC

    The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

    The Ca2+-activated protein phosphatase calcineurin induces apoptosis, but the mechanism is unknown. Calcineurin was found to dephosphorylate BAD, a pro-apoptotic member of the Bcl-2 family, thus enhancing BAD heterodimerization with Bcl-xL and promoting apoptosis. The Ca2+-induced dephosphorylation of BAD correlated with its dissociation from 14-3-3 in the cytosol and translocation to mitochondria where Bcl-xL resides. In hippocampal neurons, L-glutamate, an inducer of Ca2+ influx and calcineurin activation, triggered mitochondrial targeting of BAD and apoptosis, which were both suppressible by coexpression of a dominant-inhibitory mutant of calcineurin or pharmacological inhibitors of this phosphatase. Thus, a Ca2+-inducible mechanism for apoptosis induction operates by regulating BAD phosphorylation and localization in cells.

    Funded by: NCI NIH HHS: CA-69381; NIA NIH HHS: AG-1593; NICHD NIH HHS: HD25938; ...

    Science (New York, N.Y.) 1999;284;5412;339-43

  • Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT.

    Crabtree GR

    Department of Pathology, Stanford University Medical School, California 94062, USA.

    Cell 1999;96;5;611-4

  • Dynamic complexes of beta2-adrenergic receptors with protein kinases and phosphatases and the role of gravin.

    Shih M, Lin F, Scott JD, Wang HY and Malbon CC

    Department of Molecular Pharmacology, Diabetes & Metabolic Diseases Research Program, University Medical Center, State University of New York, Stony Brook, New York 11794-8651, USA.

    Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

    The Journal of biological chemistry 1999;274;3;1588-95

  • AKAP79 inhibits calcineurin through a site distinct from the immunophilin-binding region.

    Kashishian A, Howard M, Loh C, Gallatin WM, Hoekstra MF and Lai Y

    ICOS Corporation, Bothell, Washington 98021, USA.

    Targeting of protein kinases and phosphatases provides additional specificity to substrate selectivity in cellular signaling. In the case of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, AKAP79 has been shown to bind calcineurin and inhibit its activity in vitro (Coghlan, V., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Science 267, 108-111). In the present study, we characterized the binding regions on calcineurin A (CnA) and AKAP79 that are important for this interaction. Residues 30-98 and 311-336 on CnA, and residues 108-280 on AKAP79 were found to be important for binding. The binding of CnA by AKAP79 does not require the calcineurin B subunit, and occurs in a region distinct from where the immunosuppressant-immunophilin complex bind. AKAP79 also bound to CnA in cells transfected with AKAP79 and CnA. To determine the function of AKAP79-calcineurin interaction in intact cells, we measured the dephosphorylation and subsequent activation of NFAT, a transcription factor that is a substrate for calcineurin. Overexpression of AKAP79 inhibited NFAT dephosphorylation, resulting in a decrease in NFAT activation. These results demonstrated that AKAP79 can bind to and inhibit calcineurin activity in vivo, suggesting a physiological role for AKAP79-calcineurin interaction in NFAT-mediated signaling.

    The Journal of biological chemistry 1998;273;42;27412-9

  • Cabin 1, a negative regulator for calcineurin signaling in T lymphocytes.

    Sun L, Youn HD, Loh C, Stolow M, He W and Liu JO

    Center for Cancer Research and Deparmtent of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

    Calcineurin plays a pivotal role in the T cell receptor (TCR)-mediated signal transduction pathway and serves as a common target for the immunosuppressants FK506 and cyclosporin A. We report the identification of a novel endogenous calcineurin binding protein named Cabin 1 that inhibits calcineurin-mediated signal transduction. The interaction between Cabin 1 and calcineurin is dependent on PKC activation. Overexpression of Cabin 1 or its N-terminal truncation mutants inhibits the transcriptional activation of calcineurin-responsive elements in the interleukin-2 promoter and blocks dephosphorylation of NF-AT upon T cell activation. These results suggest a negative regulatory role for Cabin 1 in calcineurin signaling and provide a possible mechanism of feedback inhibition of TCR signaling through cross-talk between protein kinases and calcineurin.

    Immunity 1998;8;6;703-11

  • Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2.

    Shibasaki F, Kondo E, Akagi T and McKeon F

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

    It is not known how the protein Bcl-2 inhibits cell death induced by calcium signalling and growth-factor withdrawal. Here we report that Bcl-2 forms a tight complex with calcineurin, resulting in the targeting of calcineurin to Bcl-2 sites on cytoplasmic membranes, and show that this interaction is dependent on the BH4 domain of Bcl-2. Calcineurin bound to Bcl-2 is an active phosphatase but is unable to promote the nuclear translocation of NF-AT, a transcription-factor required for induction of interleukin-2 expression, suggesting a mechanism by which Bcl-2 suppresses NF-AT activity. We also show that Bax, a pro-apoptotic member of the Bcl-2 family, interferes with interactions between calcineurin and Bcl-2. We propose that the ability of Bcl-2 to block NF-AT signalling is due to the sequestering of active calcineurin to the same domain of Bcl-2 which associates with Rad-1 (ref. 5), and that calcineurin may act in Bcl-2-regulated functions.

    Nature 1997;386;6626;728-31

  • Calcineurin A alpha (PPP3CA), calcineurin A beta (PPP3CB) and calcineurin B (PPP3R1) are located on human chromosomes 4, 10q21-->q22 and 2p16-->p15 respectively.

    Wang MG, Yi H, Guerini D, Klee CB and McBride OW

    Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

    Calcineurin (also called protein phosphatase-2B) is a calmodulin-regulated protein phosphatase which plays an important role in signal transduction. The enzyme is a heterodimer of a 58-59 kDa calmodulin-binding catalytic subunit (calcineurin A) and a small (i.e. 19 kDa) Ca(2+)-binding regulatory subunit (calcineurin B). The highly conserved calcineurin B is encoded by a single gene in all tissues except testes, whereas there are three isoforms of calcineurin A (alpha, beta and gamma) encoded by genes on three different chromosomes. This enzyme can play a critical role in transcriptional regulation and growth control in T lymphocytes by a mechanism believed to involve dephosphorylation of the nuclear factor NF-AT which is essential for transcription of the interleukin-2 gene. To better evaluate the potential role of the calcineurin genes in human genetic disorders, we have studied their chromosome locations. Calcineurin B (PPP3R1) is located on human chromosome 2p16-->p15 and calcineurin A beta (PPP3CB, previous gene symbol CALNB) is present on 10q21-->q22. We confirm the localization of calcineurin A alpha (PPP3CA, previous gene symbol CALNA) to chromosome 4 without regional localization.

    Cytogenetics and cell genetics 1996;72;2-3;236-41

  • Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.

    Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW et al.

    Agouron Pharmaceuticals Inc., San Diego, California 92121-1121, USA.

    Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal struct 1e3e ures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.

    Nature 1995;378;6557;641-4

  • Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux.

    Cameron AM, Steiner JP, Roskams AJ, Ali SM, Ronnett GV and Snyder SH

    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    The immunosuppressant drug FK506 binds to the immunophilin protein FKBP12 and inhibits its prolyl isomerase activity. Immunosuppressive actions, however, are mediated via an FK506-FKBP12 inhibition of the Ca(2+)-activated phosphatase calcineurin. Physiologic cellular roles for FKBP12 have remained unclear. FKBP12 is physically associated with the RyR and IP3R Ca2+ channels in the absence of FK506, with added FK506 disrupting these complexes. Dissociation of FKBP12 results in alteration of channel Ca2+ conductance in both cases. We now report that calcineurin is physiologically associated with the IP3R-FKBP12 and RyR-FKBP12 receptor complexes and that this interaction can be disrupted by FK506 or rapamycin. Calcineurin anchored to the IP3R via FKBP12 regulates the phosphorylation status of the receptor, resulting in a dynamic Ca(2+)-sensitive regulation of IP3-mediated Ca2+ flux.

    Funded by: NIDA NIH HHS: DA-00266; NIMH NIH HHS: MH 10325-03, MH-18501; ...

    Cell 1995;83;3;463-72

  • Calcineurin functions in Ca(2+)-activated cell death in mammalian cells.

    Shibasaki F and McKeon F

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

    Calcineurin is a calcium-dependent protein phosphatase that functions in T cell activation. We present evidence that calcineurin functions more generally in calcium-triggered apoptosis in mammalian cells deprived of growth factors. Specifically, expression of epitope-tagged calcineurin A induces rapid cell death upon calcium signaling in the absence of growth factors. We show that this apoptosis does not require new protein synthesis and therefore calcineurin must operate through existing substrates. Co-expression of the Bcl-2 protooncogene efficiently blocks calcineurin-induced cell death. Significantly, we demonstrate that a calcium-independent calcineurin mutant induces apoptosis in the absence of calcium, and that this apoptotic response is a direct consequence of calcineurin's phosphatase activity. These data suggest that calcineurin plays an important role in mediating the upstream events in calcium-activated cell death.

    The Journal of cell biology 1995;131;3;735-43

  • X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex.

    Griffith JP, Kim JL, Kim EE, Sintchak MD, Thomson JA, Fitzgibbon MJ, Fleming MA, Caron PR, Hsiao K and Navia MA

    Vertex Pharmaceuticals, Incorporated, Cambridge, Massachusetts 02139-4211, USA.

    The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.

    Cell 1995;82;3;507-22

  • Association of protein kinase A and protein phosphatase 2B with a common anchoring protein.

    Coghlan VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM and Scott JD

    Vollum Institute, Oregon Health Sciences University, Portland 97201.

    Specificity of protein kinases and phosphatases may be achieved through compartmentalization with preferred substrates. In neurons, adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase (PKA) is localized at postsynaptic densities by association of its regulatory subunit with an A kinase anchor protein, AKAP79. Interaction cloning experiments demonstrated that AKAP79 also binds protein phosphatase 2B, or calcineurin (CaN). A ternary complex of PKA, AKAP, and CaN was isolated from bovine brain, and colocalization of the kinase and the phosphatase was established in neurites of cultured hippocampal neurons. The putative CaN-binding domain of AKAP79 is similar to that of the immunophilin FKBP-12, and AKAP79 inhibited CaN phosphatase activity. These results suggest that both PKA and CaN are targeted to subcellular sites by association with a common anchor protein and thereby regulate the phosphorylation state of key neuronal substrates.

    Funded by: NIDDK NIH HHS: DK09059; NIGMS NIH HHS: GM48231

    Science (New York, N.Y.) 1995;267;5194;108-11

  • Molecular cloning of a full-length cDNA encoding the catalytic subunit of human calmodulin-dependent protein phosphatase (calcineurin A alpha).

    Muramatsu T and Kincaid RL

    Section on Immunology, Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852.

    A complementary DNA for human calcineurin A alpha (protein phosphatase-2B), encoding a protein of 521 amino acids, was isolated from a hippocampus library. The deduced human sequence differs from that of mouse in only two amino acids, demonstrating that the structure of this catalytic subunit has been strictly conserved during mammalian evolution. Such high homology is in contrast to that seen for calcineurin A gamma, an isoform that shows only 88% identity between human and mouse (Muramatsu, T. and Kincaid, R.L. (1992) Biochem. Biophys. Res. Commun. 188, 265-271).

    Biochimica et biophysica acta 1993;1178;1;117-20

  • Chromosomal mapping of the human genes for the calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit.

    Giri PR, Higuchi S and Kincaid RL

    Section on Immunology, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20852.

    Multiple catalytic subunits of the Ca2+ and calmodulin (CaM)-dependent protein phosphatase (PrP) ("calcineurin" or PrP-2B) are derived from at least two structural genes, type 1 ("calcineurin A alpha") and type 2 ("calcineurin A beta "), each of which can produce alternatively spliced transcripts. To examine the possible linkage of these genes, we analyzed genomic DNA from human/hamster hybrid cell lines using probes of 122 base pairs that were designed to bind selectively to exon 3 of the open reading frame. In this region, the nucleotide sequence of the type 2 murine cDNA that we cloned was greater than 99% identical to the type 2 human cDNA but only 78% identical to the type 1 human cDNA. Hybridization to Southern blots containing DNA from all human chromosomes showed that gene 1 was found on chromosome 4, whereas gene 2 segregated to chromosome 10. These data suggest that expression of the two calcineurin genes is not physically linked.

    Biochemical and biophysical research communications 1991;181;1;252-8

  • Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods.

    Kincaid RL, Giri PR, Higuchi S, Tamura J, Dixon SC, Marietta CA, Amorese DA and Martin BM

    Section on Immunology, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852.

    The cloning and characterization of cDNAs for the catalytic subunit of calcineurin (CN) from murine and human brain libraries were carried out using nonisotopic methods. A murine cDNA clone encoding a protein of 521 amino acids (Mr approximately 58,650) was isolated; overlapping clones established a 3'-untranslated region of 554 base pairs preceding the poly(A) tail. Homologous cDNAs from human brain showed greater than 92% nucleotide sequence identity in both coding and non-coding regions with greater than 99% conservation of amino acid sequence. A second class of cDNAs lacking a specific 30-base pair region following the calmodulin-binding domain was found in four murine and human libraries. Oligonucleotide probes for both cDNA isoforms hybridized to mRNA from several brain regions indicating the existence of transcripts in vivo. The nucleotide sequences of the two forms were identical except for the inserted sequence, and Southern blot analysis of mouse and rat DNA was consistent with their having originated from the same gene; these data suggest that alternative splicing may give rise to molecular isoforms of the catalytic subunit in brain. Northern blots showed a predominant mRNA for CN in most tissues of approximately 4.0 kilobases (kb) with lower amounts of a 3.6-kb species. Brain showed 10 times more of these mRNAs than skeletal muscle while other tissues had less than or equal to 5% that in brain. In testis, multiple mRNAs were observed, with the major forms being approximately 2.8 and 1.6 kb; the total amount of CN message was about 15% that in brain. The presence of mRNA isoforms of the catalytic subunit may provide for isoenzymes of this phosphatase having distinct phosphoprotein substrate specificities or regulatory properties. The structural relatedness of CN to other mammalian serine/threonine protein phosphatases was highest over a region of approximately 240 amino acids near the amino terminus of this subunit, with greater similarity to protein phosphatase 2A than protein phosphatase 1. The conservation of many regions found in lambda phage phosphatase (Cohen, P.T.W., and Cohen, P. (1989) Biochem. J. 260, 931-934) indicates a common origin for the catalytic domain of this enzyme.

    The Journal of biological chemistry 1990;265;19;11312-9

  • Cloning of human calcineurin A: evidence for two isozymes and identification of a polyproline structural domain.

    Guerini D and Klee CB

    Laboratory of Biochemistry, National Cancer Institute, Bethesda, MD 20892.

    Two types (I and II) of cDNAs encoding the large (A) subunit of calcineurin, a calmodulin-regulated protein phosphatase, were isolated from human basal ganglia and brainstem mRNA. The complete sequences of the two calcineurin clones are identical except for a 54-base-pair insert in the type I clone and different 3' ends including part of the coding sequence for the C termini of the two proteins. These findings suggest that calcineurin A consists of at least two isozymes that may result from alternative splicing events. The two forms of the enzyme differ in the C terminus, which contains an inhibitory domain rapidly severed by limited proteolysis. With the exception of an 18-amino acid insert, the central parts of the molecules, which harbor the catalytic domains, are identical and show extended similarities with the entire catalytic subunits of protein phosphatases 1 and 2A, defining a distinct family of protein phosphatases. The 40-residue N-terminal fragment, specific for calcineurin, contains a sequence of 11 successive prolines that is also found in bovine brain calcineurin by peptide sequencing. A role in the calmodulin activation of calcineurin is proposed for this novel structural element.

    Proceedings of the National Academy of Sciences of the United States of America 1989;86;23;9183-7

  • Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin.

    Kincaid RL, Nightingale MS and Martin BM

    Section on Immunology, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892.

    A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phosphoprotein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A beta-galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3' noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain approximately 12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain.

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;23;8983-7

  • Mammalian brain phosphoproteins as substrates for calcineurin.

    King MM, Huang CY, Chock PB, Nairn AC, Hemmings HC, Chan KF and Greengard P

    Calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase found in several tissues, is highly concentrated in mammalian brain. In an attempt to identify endogenous brain substrates for calcineurin, kinetic analyses of the dephosphorylation of several well-characterized phosphoproteins purified from brain were performed. The proteins studied were: G-substrate, a substrate for cyclic GMP-dependent protein kinase; DARPP-32, a substrate for cyclic AMP-dependent protein kinase; Protein K.-F., a substrate for a cyclic nucleotide- and Ca2+-independent protein kinase; and synapsin I, a substrate for cyclic AMP-dependent (site I) and a Ca2+/calmodulin-dependent protein kinase (site II). Calcineurin dephosphorylated each of these proteins in a Ca2+/calmodulin-dependent manner. Similar Km values were obtained for each substrate: G-substrate, 3.8 microM; DARPP-32, 1.6 microM; Protein K.-F., approximately 3 microM (S0.5); synapsin I (site I), 7.0 microM; synapsin I (site II), 4.4 microM. However, significant differences were obtained for the maximal rates of dephosphorylation. The kcat values were: G-substrate, 0.41 s-1; DARPP-32, 0.20 s-1; Protein K.-F., 0.7 s-1; synapsin I (site I), 0.053 s-1; synapsin I (site II), 0.040 s-1. Comparisons of the catalytic efficiency (kcat/Km) for each substrate indicated that DARPP-32, G-substrate, and Protein K.-F. are all potential substrates for calcineurin in vivo.

    Funded by: NIMH NIH HHS: MH-17387; NINDS NIH HHS: NS-08440

    The Journal of biological chemistry 1984;259;13;8080-3

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000041 G2C Homo sapiens Pocklington H10 Human orthologues of cluster 10 (mouse) from Pocklington et al (2006) 3
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
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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