Cain, A Novel Physiologic Protein Inhibitor of Calcineurin (1998)

Calcineurin is a widely distributed protein phosphatase regulated by calcium and calmodulin. It mediates the immunosuppressive actions of drugs such as cyclosporin and FK506, and has been implicated in a number of calcium-sensitive pathways in the nervous system, including regulation of neurotransmitter release and modulation of long-term changes in synaptic plasticity. Calcineurin associates physiologically with other proteins, including calmodulin, FKBP12 (FK506-binding protein), the ryanodine receptor, and the inositol 1,4,5-trisphosphate receptor. We now report the identification, molecular cloning, and functional characterization of a novel protein, cain (calcineurininhibitor), that interacts with and inhibits calcineurin. The full-length cain cDNA predicts a 240-kDa protein with no significant homology to any known protein. Cain associates with calcineurin both in vitro and in vivo, leading to a non-competitive inhibition of calcineurin activity. The putative calcineurin-binding domain of cain, a 38-amino acid region defined by mutational analysis, is highly basic. Like calcineurin, cain has a prominent neuronal expression and a wide tissue distribution. Cain's expression pattern in the brain closely resembles that of calcineurin, indicating a physiologic association between the two proteins. Calcineurin is a widely distributed protein phosphatase regulated by calcium and calmodulin. It mediates the immunosuppressive actions of drugs such as cyclosporin and FK506, and has been implicated in a number of calcium-sensitive pathways in the nervous system, including regulation of neurotransmitter release and modulation of long-term changes in synaptic plasticity. Calcineurin associates physiologically with other proteins, including calmodulin, FKBP12 (FK506-binding protein), the ryanodine receptor, and the inositol 1,4,5-trisphosphate receptor. We now report the identification, molecular cloning, and functional characterization of a novel protein, cain (calcineurininhibitor), that interacts with and inhibits calcineurin. The full-length cain cDNA predicts a 240-kDa protein with no significant homology to any known protein. Cain associates with calcineurin both in vitro and in vivo, leading to a non-competitive inhibition of calcineurin activity. The putative calcineurin-binding domain of cain, a 38-amino acid region defined by mutational analysis, is highly basic. Like calcineurin, cain has a prominent neuronal expression and a wide tissue distribution. Cain's expression pattern in the brain closely resembles that of calcineurin, indicating a physiologic association between the two proteins. Calcineurin is a serine/threonine protein phosphatase activated by calcium and the calmodulin-calcium complex (1Stewart A.A. Ingebritsen T.S. Manalan A. Klee C.B. Cohen P. FEBS Lett. 1982; 137: 80-84Crossref PubMed Scopus (339) Google Scholar, 2Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar). This enzyme was first purified from the brain, where it was found in high concentrations in neurons (3Klee C.B. Crouch T.H. Krinks M.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6270-6273Crossref PubMed Scopus (610) Google Scholar). Calcineurin is a heterodimer consisting of a 60-kDa catalytic subunit (CnA) 1The abbreviations used are: CnA, 60-kDa catalytic subunit; CaB, 19-kDa calcium-binding regulatory subunit; NMDA, N-methyl-d-aspartate. and a 19-kDa calcium-binding regulatory subunit (CnB). The discovery that immunophilins cyclophilin and FKBP12 (FK506-binding protein) form inhibitory complexes with calcineurin in the presence of immunophilin-binding drugs such as cyclosporin A and FK506 (4Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3620) Google Scholar, 5Friedman J. Weissman I. Cell. 1991; 66: 799-806Abstract Full Text PDF PubMed Scopus (361) Google Scholar) explained the mechanism of immunosuppression by these drugs (6Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1968) Google Scholar), and established calcineurin as a mediator of T cell activation (7Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1476) Google Scholar, 8O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 9Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (755) Google Scholar). Calcineurin activity is necessary for the synthesis of several cytokine genes through the dephosphorylation of a family of transcription factors known as NF-AT (nuclear factor ofactivated T cells) (10Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (679) Google Scholar, 11Luo C. Burgeon E. Carew J.A. McCaffrey P.G. Badalian T.M. Lane W.S. Hogan P.G. Rao A. Mol. Cell. Biol. 1996; 16: 3955-3966Crossref PubMed Scopus (173) Google Scholar, 12Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1247-1274Abstract Full Text Full Text PDF Scopus (90) Google Scholar). By inhibiting calcineurin activity, cyclosporin A and FK506 prevent the nuclear translocation of NF-AT secondary to dephosphorylation, thereby suppressing T cell activation (14Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2223) Google Scholar). Although it is expressed in many tissues, calcineurin is most concentrated in neuronal systems, and comprises over 1% of total protein in brain (2Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar, 15Yakel J.L. Trends Pharmacol. Sci. 1997; 18: 124-134Abstract Full Text PDF PubMed Scopus (224) Google Scholar). Calcineurin has diverse roles in the nervous system, where it participates in many calcium-regulated pathways. Dynamin I, whose GTPase activity regulates synaptic vesicle recycling (16De Camilli P. Takei K. Neuron. 1996; 16: 2479-2487Abstract Full Text Full Text PDF Scopus (189) Google Scholar, 17Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (255) Google Scholar), is a substrate of calcineurin (18Nichols R.A. Suplick G.R. Brown J.M. J. Biol. Chem. 1994; 269: 23817-23823Abstract Full Text PDF PubMed Google Scholar, 19Liu J.P. Sim A.T. Robinson P.J. Science. 1994; 265: 970-973Crossref PubMed Scopus (183) Google Scholar). Calcineurin modulates the GTPase activity of dynamin I by altering its phosphorylation state, thereby regulating the release of neurotransmitters such as glutamate (18Nichols R.A. Suplick G.R. Brown J.M. J. Biol. Chem. 1994; 269: 23817-23823Abstract Full Text PDF PubMed Google Scholar, 20Sihra T.S. Nairn A.C. Kloppenburg P. Lin Z. Pouzat C. Biochem. Biophys. Res. Commun. 1995; 212: 609-616Crossref PubMed Scopus (44) Google Scholar). Calcineurin regulates the activity ofN-methyl-d-aspartate (NMDA) receptor channels by both altering their ion gating properties (21Lieberman D.N. Mody I. Nature. 1994; 369: 235-239Crossref PubMed Scopus (433) Google Scholar) and promoting desensitization (22Tong G. Shepherd D. Jahr C.E. Science. 1995; 267: 1510-1512Crossref PubMed Scopus (353) Google Scholar). Elevation of intracellular calcium concentration following opening of the NMDA receptor channel activates several downstream proteins including neuronal nitric oxide synthase, which influences neuronal cell death secondary to glutamate NMDA neurotoxicity (23Dawson V.L. Bredt D.S. Fotuhi M. Hwang P.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7797-7801Crossref PubMed Scopus (1840) Google Scholar, 24Choi D.W. Science. 1992; 258: 241-243Crossref PubMed Scopus (164) Google Scholar). The activity of nitric oxide synthase is diminished by phosphorylation so that dephosphorylation and subsequent activation of nitric oxide synthase by calcineurin implicates this phosphatase in NMDA-mediated neurotoxicity (25Dawson T.M. Steiner J.P. Dawson V.L. Dinerman J.L. Uhl G.R. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9808-9812Crossref PubMed Scopus (512) Google Scholar). Besides mediating glutamate neurotoxicity, NMDA receptors influence synaptic plasticity. A role for protein kinases in shaping synaptic plasticity is well established (26Roberson E.D. English J.D. Sweatt J.D. Learn. Mem. 1996; 3: 1-24Crossref PubMed Scopus (99) Google Scholar), as multiple experimental paradigms have demonstrated a close correlation between reduction of kinase activity and impairment of long-term potentiation in the hippocampus (27Abel T. Nguyen P.V. Barad M. Deuel T.A.S. Kandel E.R. Bourtchouladze R. Cell. 1997; 88: 1-11Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 28Huang Y.-Y. Nguyen P.V. Abel T. Kandel E.R. Learn. Mem. 1996; 3: 74-85Crossref PubMed Scopus (197) Google Scholar, 29Mayford M. Mansuy I.M. Muller R.U. Kandel E.R. Curr. Biol. 1997; 7: R580-R589Abstract Full Text Full Text PDF PubMed Google Scholar). More recently, phosphatases, especially calcineurin, have been implicated in regulating synaptic plasticity (15Yakel J.L. Trends Pharmacol. Sci. 1997; 18: 124-134Abstract Full Text PDF PubMed Scopus (224) Google Scholar). Calcineurin has diverse, sometimes contradictory, effects on synaptic plasticity (30Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (905) Google Scholar, 31Lu Y.F. Hayashi Y. Moriwaki A. Tomizawa K. Matsui H. Neurosci. Lett. 1996; 205: 103-106Crossref PubMed Scopus (33) Google Scholar, 32Wang J.H. Kelly P.T. J. Neurosci. 1997; 17: 4600-4611Crossref PubMed Google Scholar). Recently, Winder and colleagues (33Winder D.G. Mansuy I.M. Osman M. Moallem T.M. Kandel E.R. Cell. 1998; 92: 25-37Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) discovered that calcineurin inhibits a novel, intermediate phase of long-term potentiation, which influences the transition from short-term to long-term memory (34Mansuy I.M. Mayford M. Jacob B. Kandel E.R. Bach M.E. Cell. 1998; 92: 39-49Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Despite the abundance of calcineurin and its role in many biologically important processes, few endogenous regulators of calcineurin have been identified. Calmodulin, bound to CnA, enhances its activity, while the autoinhibitory domain of CnA competitively inhibits the phosphatase activity (35Hashimoto Y. Perrino B.A. Soderling T.R. J. Biol. Chem. 1990; 265: 1924-1947Abstract Full Text PDF PubMed Google Scholar). Superoxide dismutase prevents the in vivoinactivation of calcineurin secondary to oxidative damage (36Wang X. Culotta V.C. Klee C.B. Nature. 1996; 383: 434-437Crossref PubMed Scopus (241) Google Scholar), but there is no evidence for direct binding between the two proteins. FKBP12 associates with calcineurin, even in the absence of immunosuppressive drugs (37Cardenas M.E. Hemenway C. Muir R.S. Ye R. Fiorentino D. Heitman J. EMBO J. 1994; 13: 5944-5957Crossref PubMed Scopus (138) Google Scholar). This interaction involves residues distinct from those mediating the FK506-dependent association of FKBP12 and calcineurin, and allows FKBP12 to function as a calcineurin anchoring protein. FKBP12 targets calcineurin to intracellular calcium channels such as the ryanodine receptor (38Jayaraman T. Brillantes A.M. Timerman A.P. Fleischer S. Erdjument-Bromage H. Tempst P. Marks A.R. J. Biol. Chem. 1992; 267: 9474-9477Abstract Full Text PDF PubMed Google Scholar, 39Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar, 40Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondrisova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (703) Google Scholar) and the inositol 1,4,5-trisphosphate receptor (41Cameron A.M. Steiner J.P. Sabatini D.M. Kaplin A.I. Walensky L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1784-1788Crossref PubMed Scopus (269) Google Scholar). Calcineurin activity regulates the calcium flux through these channels secondary to changes in their phosphorylation states (42Cameron A.M. Steiner J.P. Roskams A.J. Ali S.M. Ronnett G.V. Snyder S.H. Cell. 1995; 83: 463-472Abstract Full Text PDF PubMed Scopus (451) Google Scholar). Another well known anchoring protein for calcineurin is AKAP79 (Akinase anchoring protein), a scaffolding protein that also binds cAMP-dependent protein kinase and protein kinase C (43Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (482) Google Scholar). Physical association between calcineurin and AKAP79 leads to inhibition of calcineurin activity, as well as its targeting to neuronal membranes (44Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (529) Google Scholar). It is unlikely that the above short list of proteins could adequately explain the diversity of calcineurin function. To better appreciate the role of calcineurin in multiple calcium-sensitive signaling pathways, we sought additional regulatory molecules for this unique phosphatase. We now report the identification and characterization of cain (calcineurin inhibitor), a novel, large protein that physiologically interacts with calcineurin and potently inhibits its catalytic activity. FK506 was a gift from Dr. Joseph Steiner (Guilford Pharmaceutical). Glutathione-Sepharose and calmodulin-Sepharose beads were purchased from Pharmacia Biotech. Purified human FKBP12, purified bovine calcineurin, the RII peptide (calcineurin substrate), and cAMP-dependent protein kinase were purchased from Sigma. Anti-CnA antibody was obtained from Transduction Laboratories, and anti-CnB antibody was purchased from Upstate Biotechnology Inc. The rabbit polyclonal anti-cain antibody was raised against (His)6-cain (described below), and affinity-purified using the immunogen as described (45Sharp A.H. McPherson P.S. Dawson T.M. Aoki C. Campbell K.P. Snyder S.H. J. Neurosci. 1993; 13: 3051-3063Crossref PubMed Google Scholar). CnA was subcloned into SalI andNotI sites of pPC86/97 (46Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (480) Google Scholar) from the full-length human calcineurin Aβ open using the following and Cain were from the cain obtained from the using the following and were with SalI and and into sites in and Pharmacia of the were by The full-length human FKBP12 open was into expression the This was used to a cDNA into S.H. A.H. Jr., G. A. P. Snyder S.H. Nature. 1995; PubMed Scopus Google Scholar), the The were by into as described U. S. T. J. J.H. Res. 1996; PubMed Scopus Google Scholar). A total of were and proteins were by for with a on the presence of were for expression by as described (46Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (480) Google Scholar). proteins were to and to (His)6-cain was by the cain from the into the SalI and sites of The protein was as the binding of purified (His)6-cain of purified bovine calcineurin were to of the in and to a of with binding bovine binding was to for with the were with binding in and by were and with antibody and anti-CnB antibody by a of antibody to were using The cain was subcloned into a expression with a and This was into by calcium were following and in of cell were with calmodulin-Sepharose beads of for with beads were with FK506 for and in to to the cell FK506 was also to the cell in as the the beads were with and in and were as described The antibody was used and antibody brain was in (described and for to The was into and with beads in the described proteins were by and as brain cDNA in was using the cain with in a A open was and a from the was for a A total of were of cDNA was using brain cDNA to The was as described by Hubbard and Klee M.J. Klee C.B. H. J. Scholar) A peptide to the phosphorylation on the RII subunit of cAMP-dependent protein kinase was and used as were in bovine calmodulin, and bovine calcineurin with (His)6-cain were by the of substrate were for and by the of acid in A multiple tissue was to the with the cain described from were and in and was for and the of from were by and was using affinity-purified polyclonal anti-cain by and with for FKBP12, CnA, and cain were by open from into the SalI and sites of for in were and as described S. Snyder S.H. J. Neurosci. 1997; 17: PubMed Google Scholar). we the to proteins that FKBP12 in the presence of the We a cDNA and a acid protein homology to any known protein This the of cain the analysis, and were used to protein the activation of two expression and the endogenous FKBP12 we a interaction between FKBP12 and CnA in the presence of FK506 and We that the endogenous FKBP12 with the human FKBP12 protein in the we the interaction between FKBP12 and CnA in the and a interaction and of in in of FK506 of of the and the The was by a of to to into the SalI and sites of in a the and the The was by a of to to into the SalI and sites of the we found that cain with FKBP12 in the presence of FK506 I, the of this interaction that the association between cain and by a protein. calcineurin binds to FKBP12 in the presence of FK506, proteins that with calcineurin as regulators with CnA as the protein in the system, we found a interaction with cain I, The of this interaction was in the absence of FK506 and endogenous FKBP12, indicating a direct association between calcineurin and purified proteins a direct interaction between cain and calcineurin. purified (His)6-cain bound in the presence of calcineurin, that there is no direct interaction between cain and FKBP12 This is by binding and purified calcineurin. The direct association between calcineurin and cain was by the of FK506 and FKBP12 To the interaction between cain and calcineurin in we with We of and the endogenous calcineurin a and calcineurin bound the in the presence of The interaction between and calcineurin also allows for of calcineurin by a this we the of to with calcineurin the presence of calmodulin-Sepharose but both and calcineurin bound the in a We cain and calcineurin physiologically in brain we bound endogenous calcineurin and for cain proteins using anti-cain The of was in to association of endogenous proteins, secondary that We found that cain and calcineurin in the presence of FK506, and that the of cain in with the of bound calcineurin The association between cain and calcineurin could also in tissue this that the endogenous cain protein has molecular of from the cain we a brain cDNA and obtained A of cDNA no additional The full-length cDNA of cain from The was to the first This is by two and in the of a M. J. Biol. PubMed Scopus Google Scholar). The open for a protein of with a molecular of A of no significant homology with any protein. To the region of cain that interacts with calcineurin, we by the in the the system, we found two of cain that the to with calcineurin This was by in vitro binding using purified calcineurin and cain to beads that of the calcineurin binding activity of cain to a 38-amino acid region the C This is in that the for this 38-amino acid is while that of the full-length is To influences of cain on calcineurin catalytic activity, we calcineurin activity in the presence of purified Cain potently calcineurin vitro with inhibition and a of A that the inhibition is non-competitive To the that the inhibition of calcineurin activity by cain explained by its binding to calmodulin, we inhibition in the presence of concentrations of calmodulin. We found that the inhibition of calcineurin by cain is of in the presence of a in the inhibition was analysis, we a of cain in brain, and but which also in other This is with full-length a and from in using purified polyclonal anti-cain antibody a The of this on is with the molecular of full-length were also prominent in and of the full-length protein. of these were by the antibody with (His)6-cain expression closely the by analysis, in to the abundance of cain in and that cain is Calcineurin and FKBP12 highly in the brain, a functional interaction between these two proteins T.M. Steiner J.P. Fotuhi M. M. Snyder S.H. 1994; PubMed Scopus Google Scholar). of cain, calcineurin, and FKBP12, a between cain and calcineurin expression expression in the but prominent in the of the and of the hippocampus and The of cain and calcineurin even that of FKBP12 and calcineurin. the where FKBP12 is both cain and calcineurin in abundance FKBP12 is to and to cain and calcineurin both in calcineurin also in the we have a novel protein, cain, which physiologically interacts with and inhibits calcineurin. experimental paradigms including the and several binding that the interaction vitro and in We also physiologic association of cain and calcineurin in both brain and the most evidence a physiologic between cain and calcineurin is their in the brain, where both proteins in we a close between of calcineurin and in the brain, a between the two proteins T.M. Steiner J.P. Fotuhi M. M. Snyder S.H. 1994; PubMed Scopus Google even between cain and calcineurin expression that between calcineurin and Cain inhibits calcineurin with a of and to the most known endogenous of calcineurin. a well established calcineurin is as in this (44Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (529) Google Scholar). The autoinhibitory peptide of calcineurin has of (35Hashimoto Y. Perrino B.A. Soderling T.R. J. Biol. Chem. 1990; 265: 1924-1947Abstract Full Text PDF PubMed Google Scholar). the inhibitory of cain is CnA is we binding of cain to both CnA and the two calcineurin so that it is that as a Cain binds to calcineurin distinct from the as multiple that the association between cain and calcineurin is by the of to the of calcineurin, thereby inhibiting calcineurin activity A. X. R. Nature. 1995; PubMed Scopus Google Scholar, J.P. J.L. J.A. M.J. M.A. K. M.A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). The binding and subsequent inhibition of calcineurin by cain regulatory on calcineurin. We have that this is most distinct from the domain of calcineurin, as changes in concentration no in inhibition of calcineurin activity. cain physiologically that cain is a protein. it could well to the to which it Cain function as a scaffolding protein to proteins calcineurin. with this is the that calcineurin binding to cain is to a in the of the protein. of cain in to large protein that binds and inhibits calcineurin. both cain and the putative calcineurin-binding a of acid residues (44Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (529) Google Scholar). AKAP79 binds cAMP-dependent protein kinase and protein kinase C as well as calcineurin. cain could also the kinase and phosphatase of proteins to cain cain is as phosphorylation for cain have been Although we have cain as of calcineurin, its role to a for calcineurin in its this cain calcineurin to intracellular where its release calcium-regulated phosphatase activity to signaling pathways. We and for and of the We also for the full-length cain Cohen for with multiple tissue Dr. Klee for the gift of human Dr. Joseph Steiner for a gift of FK506, Sabatini and of the Snyder for and