Regulation of the Calmodulin-stimulated Protein Phosphatase, Calcineurin
1998; Elsevier BV; Volume: 273; Issue: 22 Linguagem: Inglês
10.1074/jbc.273.22.13367
ISSN1083-351X
AutoresClaude B. Klee, Hao Ren, Xutong Wang,
Tópico(s)Nerve injury and regeneration
ResumoThe role of protein phosphatases in the regulation of cellular processes is now well established (1Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (403) Google Scholar, 2Cohen P.T. Chen M.X. Armstrong C.G. Adv. Pharmacol. 1996; 36: 67-89Crossref PubMed Scopus (37) Google Scholar). Calcineurin (also called protein phosphatase 2B), a major calmodulin-binding protein in brain and the only serine/threonine protein phosphatase under the control of Ca2+/calmodulin, plays a critical role in the coupling of Ca2+ signals to cellular responses (3Klee C.B. Crouch T.H. Krinks M.H. Proc. Natl. Acad. Sci. U. S. A. 1980; 76: 6270-6273Crossref Scopus (610) Google Scholar, 4Stewart A.A. Ingebritsen T.S. Manalan A. Klee C.B. Cohen P. FEBS Lett. 1982; 137: 80-84Crossref PubMed Scopus (339) Google Scholar, 5Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar, 6Kincaid R.L. Adv. Second Messenger Phosphoprotein Res. 1993; 27: 1-25PubMed Google Scholar). Its stimulation by the multifunctional protein, calmodulin, ensures the coordinated regulation of its protein phosphatase activity with the activities of the many other enzymes, including a large number of protein kinases, under Ca2+ and calmodulin control. Despite its special abundance in neural tissues, calcineurin is broadly distributed, and its structure is highly conserved from yeast to man (6Kincaid R.L. Adv. Second Messenger Phosphoprotein Res. 1993; 27: 1-25PubMed Google Scholar). Its resistance to the endogenous phosphatase inhibitor 1 and inhibitor 2 and to the potent inhibitors of protein phosphatase 1 and 2A, okadaic acid, calyculin, and microcystin (1Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (403) Google Scholar, 2Cohen P.T. Chen M.X. Armstrong C.G. Adv. Pharmacol. 1996; 36: 67-89Crossref PubMed Scopus (37) Google Scholar) made it difficult to identify its functions until it was identified as the target of the immunosuppressive drugs, FK506 and cyclosporin A (CsA). 1The abbreviations used are: CsA, cyclosporin A; IP3, inositol trisphosphate; FKBP, FK506-binding protein; NMDA, N-methyl-d-aspartate.Calcineurin was thus shown to play an essential role in T cell activation (7Liu J. Farmer 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). The demonstration that FK506 and CsA, when bound to their respective binding proteins, FKBP12 and cyclophilin A, are specific inhibitors of calcineurin provided the tools needed to reveal its many other roles in the transduction of Ca2+ signals (8Liu J. Albers M.W. Wandless T.J. Luan S. Alberg D.G. Belshaw P.J. Cohen P. MacKintosh C. Klee C.B. Schreiber S.L. Biochemistry. 1992; 31: 3896-3901Crossref PubMed Scopus (502) Google Scholar). Its calmodulin dependence distinguishes it from two other known Ca2+-regulated protein phosphatases, the insulin-sensitive pyruvate dehydrogenase phosphatase of mitochondria (9Denton R.M. McCormack J.G. Rutter G.A. Burnett P. Edgell N.J. Moule S.K. Diggle T.A. Adv. Enzyme Regul. 1996; 36: 183-198Crossref PubMed Scopus (54) Google Scholar) and a family of protein phosphatases homologous to the product of the Drosophila retinal degeneration C (rdgC) gene (10Steele F.R. Washburn T. Rieger R. O'Tousa J.E. Cell. 1992; 69: 669-676Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 11Vinos J. Jalink K. Hardy R.W. Britt S.G. Zuker C.S. Science. 1997; 277: 687-690Crossref PubMed Scopus (103) Google Scholar, 12Sherman P.M. Sun H. Macke J.P. Williams J. Smallwood P.M. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11639-11644Crossref PubMed Scopus (50) Google Scholar). Calcineurin has a relatively narrow substrate specificity. Phosphoproteins listed in Table I are preferentially dephosphorylated by calcineurin whereas others such as casein, synapsin 1, and calmodulin kinase II are dephosphorylated at much slower rates or not at all (5Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar). Other potentially physiological substrates, whose kinetic characteristics have not been determined, include NO synthase, a GTPase involved in endocytosis (dynamin, previously called dephosphin), the transcription factor Elk-1, and the heat shock protein, hsp25 (13Dawson 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, 14Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 15Sugimoto T. Stewart S. Guan K.-L. J. Biol. Chem. 1997; 272: 29415-29418Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 16Gaestel M. Benndorf R. Hayess K. Priemer E. Engel K. J. Biol. Chem. 1992; 267: 21607-21611Abstract Full Text PDF PubMed Google Scholar). The substrate specificity of calcineurin is not due only to a specific sequence but rather is determined by both primary and higher order structural features (17Blumenthal D.K. Takio K. Hansen R.S. Krebs E.G. J. Biol. Chem. 1985; 261: 8140-8145Abstract Full Text PDF Google Scholar,18Donella-Deana A. Krinks M.H. Ruzzene M. Klee C.B. Pinna L.A. Eur. J. Biochem. 1994; 219: 109-117Crossref PubMed Scopus (73) Google Scholar). The phosphorylation-independent tight binding of substrates, such as described for the transcription factor NF-ATp (nuclear factor-activated T cells), may allow calcineurin to dephosphorylate proteins whose intracellular concentration is very low (19Jain 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, 20Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1274-1277Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Calcineurin also dephosphorylates phosphotyrosine, but theK cat, except when determined in the presence of Ni2+, is 2 orders of magnitude lower than that for phosphoserine (Table I).Table ISubstrate specificity of calcineurinSubstratesk catk mRef.s−1mmInhibitor 1aValues of k cat measured in the absence of added metal are underestimated.2.80.0035Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google ScholarRII subunit2.70.0217Blumenthal D.K. Takio K. Hansen R.S. Krebs E.G. J. Biol. Chem. 1985; 261: 8140-8145Abstract Full Text PDF Google ScholarNeurogranin1.80.01393Seki K. Chen H.C. Huang K.-P. Arch. Biochem. Biophys. 1995; 316: 673-679Crossref PubMed Scopus (68) Google ScholarPhosphorylase kinase (α subunit)aValues of k cat measured in the absence of added metal are underestimated.1.40.0065Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google ScholarNeuromodulin0.1–0.50.01593Seki K. Chen H.C. Huang K.-P. Arch. Biochem. Biophys. 1995; 316: 673-679Crossref PubMed Scopus (68) Google ScholarMicrotubule-associated proteinsMAP-20.6–2.20.0025Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google ScholarTau factor0.6–0.80.0025Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google ScholarDARPP-32aValues of k cat measured in the absence of added metal are underestimated.0.20.0145Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google ScholarDLDVPIPGRFDRRVSVAAEbSynthetic peptide routinely used for calcineurin assays. Values of 12 s−1 and 0.1 mm have been reported recently (62, 95). For comparison at pH 7.5 in the absence of added metal the k cat value for the dephosphorylation of the peptide substrate by crude calcineurin is 40 s−1.2.20.02694Blumenthal D.K. Chan C.P. Takio K. Gallis B. Hansen R.S. Krebs E.G. Adv. Protein Phosphatases. 1985; 1: 163-171Google Scholar5.9cAssayed in the presence of 1 mmNi2+.0.02394Blumenthal D.K. Chan C.P. Takio K. Gallis B. Hansen R.S. Krebs E.G. Adv. Protein Phosphatases. 1985; 1: 163-171Google ScholarDLDVPIPGRFDRRVYVAAE<0.0294Blumenthal D.K. Chan C.P. Takio K. Gallis B. Hansen R.S. Krebs E.G. Adv. Protein Phosphatases. 1985; 1: 163-171Google Scholar0.3cAssayed in the presence of 1 mmNi2+.0.00394Blumenthal D.K. Chan C.P. Takio K. Gallis B. Hansen R.S. Krebs E.G. Adv. Protein Phosphatases. 1985; 1: 163-171Google Scholarp-Nitrophenyl phosphate2623.095Mertz P. Yu L. Sikkink R. Rusnak F. J. Biol. Chem. 1997; 272: 21296-21302Abstract Full Text Full Text PDF PubMed Scopus (63) Google ScholarExcept when indicated kinetic constants were measured in the presence of Mn2+ or Mg2+.a Values of k cat measured in the absence of added metal are underestimated.b Synthetic peptide routinely used for calcineurin assays. Values of 12 s−1 and 0.1 mm have been reported recently (62Etzkorn F.A. Chang Z.Y. Stolz L.A. Walsh C.T. Biochemistry. 1994; 33: 2380-2388Crossref PubMed Scopus (69) Google Scholar, 95Mertz P. Yu L. Sikkink R. Rusnak F. J. Biol. Chem. 1997; 272: 21296-21302Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For comparison at pH 7.5 in the absence of added metal the k cat value for the dephosphorylation of the peptide substrate by crude calcineurin is 40 s−1.c Assayed in the presence of 1 mmNi2+. Open table in a new tab Except when indicated kinetic constants were measured in the presence of Mn2+ or Mg2+. The synthetic peptide corresponding to residues 81–99 of the RII subunit of cAMP-dependent protein kinase (Table I) is most commonly used to measure calcineurin phosphatase activity (17Blumenthal D.K. Takio K. Hansen R.S. Krebs E.G. J. Biol. Chem. 1985; 261: 8140-8145Abstract Full Text PDF Google Scholar). Because it is a poor substrate for protein phosphatases 1, 2A, and 2C it is well suited to quantitate the Ca2+/calmodulin-dependent, metal-independent, okadaic acid-insensitive calcineurin activity in crude tissue extracts provided that the incubation time is reduced to 1–2 min to minimize Ca2+-dependent calcineurin inactivation (21Hubbard M.J. Klee C.B. Chad J. Wheal H. Molecular Neurobiology: A Practical Approach. Oxford University Press, Oxford1991: 135-157Google Scholar, 22Stemmer P.M. Wang X. Krinks M.H. Klee C.B. FEBS Lett. 1995; 374: 237-240Crossref PubMed Scopus (37) Google Scholar, 23Wang X. Culotta V.C. Klee C.B. Nature. 1996; 383: 434-437Crossref PubMed Scopus (241) Google Scholar, 24Fruman D.A. Pai S.-Y. Klee C.B. Burakoff S.J. Bierer B.E. Methods: A Companion to Methods Enzymol. 1996; 9: 146-154Crossref Scopus (64) Google Scholar). The conveniently measurablep-nitrophenyl-phosphatase activity has been employed to study its catalytic mechanism and to propose a catalysis involving the protonation of the phosphoester bond by a metal-activated water molecule followed by the cleavage of the bond by a second metal-activated water molecule, without the formation of a covalent intermediate (25Martin B.L. Graves D.J. Biochim. Biophys. Acta. 1994; 1206: 136-142Crossref PubMed Scopus (50) Google Scholar). This mechanism is consistent with the metal requirement for calcineurin activity (5Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar), the identification of calcineurin as an iron-zinc enzyme (26King M.M. Huang C.Y. J. Biol. Chem. 1984; 259: 8847-8854Abstract Full Text PDF PubMed Google Scholar), and the demonstration that calcineurin contains a binuclear [Fe3+-Zn2+] metal center (27Yu L. Haddy A. Rusnak F. J. Am. Chem. Soc. 1995; 117: 10147-10148Crossref Scopus (59) Google Scholar). In the recently published crystal structures of calcineurin (28Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (772) Google Scholar, 29Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Gastinel L.N. Habuka N. Chen X. Maldonado F. Barker J.E. Bacquet R. Villafranca J.E. Nature. 1995; 378: 641-644Crossref PubMed Scopus (697) Google Scholar), these two metal ions are modeled on the structure of the [Fe3+-Zn2+] kidney bean purple acid phosphatase. The high specific activity of calcineurin in crude extracts in the absence of added metals suggests that the crude enzyme has retained its natural cofactors (22Stemmer P.M. Wang X. Krinks M.H. Klee C.B. FEBS Lett. 1995; 374: 237-240Crossref PubMed Scopus (37) Google Scholar). Inactivation of crude calcineurin by the superoxide anion and its protection and reactivation by ascorbate strongly suggest that reduced iron is required for activity (23Wang X. Culotta V.C. Klee C.B. Nature. 1996; 383: 434-437Crossref PubMed Scopus (241) Google Scholar). Regardless of its source, calcineurin is always a heterodimer of a 58–64-kDa catalytic and calmodulin-binding subunit, calcineurin A, tightly bound (even in the presence of only nanomolar concentrations of Ca2+) to a regulatory, 19-kDa Ca2+-binding regulatory subunit, calcineurin B (5Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar). This two-subunit structure, unique among the protein phosphatases, is conserved from yeast to man and is essential for activity. Also highly conserved are the amino acid sequences of the catalytic and regulatory domains of calcineurin A isoforms from different organisms (2Cohen P.T. Chen M.X. Armstrong C.G. Adv. Pharmacol. 1996; 36: 67-89Crossref PubMed Scopus (37) Google Scholar, 6Kincaid R.L. Adv. Second Messenger Phosphoprotein Res. 1993; 27: 1-25PubMed Google Scholar). The primary structure of the α, β, and γ isoforms of mammalian calcineurin A, products of three different genes, 2The gene symbols are PPP3CAa, PPP3CAb, PPP3R1, and PPP3R2 for human calcineurin Aα, calcineurin Aβ, calcineurin B1, and calcineurin B2, respectively. is shown in Fig. 1. With the exception of variable N- and C-terminal tails, whose functions are not known, the three enzymes exhibit 83–89% identity over the remaining 90% of their sequence. An N-terminal polyproline motif is a conserved feature of the β isoform, whereas several additional basic residues in the C-terminal tail are responsible for the high pI (7.1) of the testis-specific γ-isoform, as opposed to pIs of 5.6 and 5.8 for the neural α and the broadly distributed β isoforms (5Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar, 6Kincaid R.L. Adv. Second Messenger Phosphoprotein Res. 1993; 27: 1-25PubMed Google Scholar, 30Goto S. Matsukado Y. Mihara Y. Inoue N. Miyamoto E. Acta Neuropathol. 1986; 72: 150-156Crossref PubMed Scopus (33) Google Scholar, 31Kuno T. Mukai H. Ito A. Chang C.D. Kishima K. Saito N. Tanaka C. J. Neurochem. 1992; 58: 1643-1651Crossref PubMed Scopus (126) Google Scholar). Calcineurin B is a highly conserved protein originally identified as an "EF-hand" Ca2+-binding protein on the basis of its amino acid sequence (32Aitken A. Klee C.B. Cohen P. Eur. J. Biochem. 1984; 139: 663-671Crossref PubMed Scopus (116) Google Scholar). Its dumbbell structure, determined by multidimensional NMR, is similar to that of calmodulin with two lobes, each composed of two adjacent Ca2+-binding loops connected by a flexible helix linker (33Anglister J. Grzesiek S. Wang A.C. Ren H. Klee C.B. Bax A. Biochemistry. 1994; 33: 3540-3547Crossref PubMed Scopus (48) Google Scholar). As predicted from its sequence, it binds 4 mol of Ca2+, one with high affinity (k d < 10−7m) and three with affinities in the micromolar range (34Kakalis L.T. Kennedy M. Sikkink R. Rusnak F. Armitage I.M. FEBS Lett. 1995; 362: 55-58Crossref PubMed Scopus (50) Google Scholar). Equally conserved from yeast to man is the myristoylation of the N-terminal glycine (35Aitken A. Cohen P. Santikarn S. Williams D.H. Calder A.G. Smith A. Klee C.B. FEBS Lett. 1982; 150: 314-317Crossref PubMed Scopus (180) Google Scholar, 36Cyert M.S. Thorner J. Mol. Cell. Biol. 1992; 12: 3460-3469Crossref PubMed Scopus (207) Google Scholar). There are two mammalian isoforms of calcineurin B,2 CNB1 originally found associated with calcineurin Aα and β and CNB2, which is expressed only in testis; only one form has been reported in fruit flies and the budding yeast (6Kincaid R.L. Adv. Second Messenger Phosphoprotein Res. 1993; 27: 1-25PubMed Google Scholar). The highly conserved multidomain structure of calcineurin A, illustrated in Fig. 1, was first revealed by limited proteolysis (37Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (169) Google Scholar). The catalytic domain (residues 70–328 of calcineurin Aα), followed by the calcineurin B-binding domain localized by site-directed mutagenesis and binding of synthetic peptides to calcineurin B between residues 333 and 390, is resistant to proteolysis (37Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (169) Google Scholar, 38Husi H. Luyten M.A. Zurini M.G. J. Biol. Chem. 1994; 269: 14199-14204Abstract Full Text PDF PubMed Google Scholar, 39Clipstone N.A. Fiorentino D.F. Crabtree G.R. J. Biol. Chem. 1994; 269: 26431-26437Abstract Full Text PDF PubMed Google Scholar, 40Milan D. Griffith J. Su M. Roydon Price E. McKeon F. Cell. 1994; 79: 437-447Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 41Anglister J. Ren H. Klee C.B. Bax A. FEBS Lett. 1995; 375: 108-112Crossref PubMed Scopus (9) Google Scholar, 42Sikkink R. Haddy A. MacKelvie S. Mertz P. Litwiller R. Rusnak F. Biochemistry. 1995; 34: 8348-8356Crossref PubMed Scopus (40) Google Scholar, 43Watanabe Y. Perrino B.A. Chang B.H. Soderling T.R. J. Biol. Chem. 1995; 270: 456-460Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These domains, still associated with calcineurin B, are often referred to as the Ca2+-independent form of calcineurin. The enzymatic activity of calcineurin is repressed in the native protein, but it becomes fully active when severed by proteases from the regulatory domain (residues 390–521). The regulatory domain, readily susceptible to proteolysis in the absence of calmodulin, contains two subdomains: a calmodulin-binding and an autoinhibitory subdomain (37Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (169) Google Scholar, 44Kincaid R.L. Martin B.M. Adv. Exp. Med. Biol. 1989; 255: 347-358Crossref PubMed Scopus (5) Google Scholar, 45Hashimoto Y. Perrino B.A. Soderling T.R. J. Biol. Chem. 1990; 265: 1924-1927Abstract Full Text PDF PubMed Google Scholar). The crystal structures of the recombinant α isoform of human calcineurin and of its complex with FKBP12-FK506 3No changes in the structure of the recombinant calcineurin were detected upon complex formation with FKBP-FK506 (29Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Gastinel L.N. Habuka N. Chen X. Maldonado F. Barker J.E. Bacquet R. Villafranca J.E. Nature. 1995; 378: 641-644Crossref PubMed Scopus (697) Google Scholar). (29Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Gastinel L.N. Habuka N. Chen X. Maldonado F. Barker J.E. Bacquet R. Villafranca J.E. Nature. 1995; 378: 641-644Crossref PubMed Scopus (697) Google Scholar) and that of the complex with FKBP12-FK506 (28Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (772) Google Scholar) of the proteolytic fragment of bovine calcineurin, lacking the regulatory domain and the N-terminal 16 residues, have been determined at 2.1, 3.5, and 2.5 Å, respectively. With the exception of the N-terminal residues 1–16 and the regulatory domain of calcineurin A, missing in the bovine protein, the crystal structure of the Ca2+-saturated form of the truncated bovine calcineurin shown in Fig. 2 is similar to that of the full-length recombinant protein. The catalytic domain, similar to that of protein phosphatase 1 (46Barford D. Trends Biochem. Sci. 1996; 21: 407-417Abstract Full Text PDF PubMed Scopus (315) Google Scholar), consists of a sandwich of a sheet of six β strands covered by three α helices and three β strands and a sheet of five β strands covered by an all helical structure. The two metal ions, iron and zinc, bound to residues provided by the two faces of the β sandwich, define the catalytic center. The last β sheet extends into a five-turn amphipathic α helix (residues 350–370) whose top face, completely non-polar, is covered by a 33-Å groove formed by the N- and C-terminal lobes and the C-terminal strand of calcineurin B. The contacts between the two subunits are in good agreement with the regions of calcineurin B involved in the interaction with calcineurin A (linkers between helix 1 and the Ca2+-binding loop 1, Ca2+-binding loops 3 and 4, the central helix linker, and the C-terminal tail) determined in solution (40Milan D. Griffith J. Su M. Roydon Price E. McKeon F. Cell. 1994; 79: 437-447Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 41Anglister J. Ren H. Klee C.B. Bax A. FEBS Lett. 1995; 375: 108-112Crossref PubMed Scopus (9) Google Scholar). Interaction of residues 14–23 of calcineurin A with the C-terminal lobe of calcineurin B may provide the additional binding energy to account for the very high affinity of calcineurin B for calcineurin A (k d < 10−13m) 4H. Ren, X. Wang, and C. Klee, unpublished observations. as opposed to the relatively low affinity of the calcineurin B-binding peptides of calcineurin A for calcineurin B (41Anglister J. Ren H. Klee C.B. Bax A. FEBS Lett. 1995; 375: 108-112Crossref PubMed Scopus (9) Google Scholar, 43Watanabe Y. Perrino B.A. Chang B.H. Soderling T.R. J. Biol. Chem. 1995; 270: 456-460Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In the bovine protein, myristic acid, covalently linked to the N-terminal glycine of calcineurin B, lies parallel to the hydrophobic face of the N-terminal helix of calcineurin B whereas the non-myristoylated N terminus of the recombinant protein is disordered. This perfectly conserved post-translational modification of calcineurin B, apparently not involved in membrane association, is not required for activity but may serve as a stabilizing structural element (47Zhu D. Cardenas M.E. Heitman J. J. Biol. Chem. 1995; 270: 24831-24838Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 48Kennedy M.T. Brockman H. Rusnak F. J. Biol. Chem. 1996; 271: 26517-26521Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the full-length protein, with the exception of two short α helices corresponding to the inhibitory domain that block the catalytic center, the regulatory domain is not visible in the electron density map (29Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Gastinel L.N. Habuka N. Chen X. Maldonado F. Barker J.E. Bacquet R. Villafranca J.E. Nature. 1995; 378: 641-644Crossref PubMed Scopus (697) Google Scholar). The disordered structure of this domain is consistent with its extreme sensitivity to proteolytic attack (37Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (169) Google Scholar). The polar bottom face of the calcineurin B-binding helix of calcineurin A, exposed to solvent, constitutes, together with calcineurin B, the binding domain of the FK506-FKBP12 complex. FKBP12 interacts with calcineurin B, the catalytic and the calcineurin B-binding domain of calcineurin A, whereas the interface of the calcineurin B-binding domain of calcineurin A and calcineurin B forms the binding site of FK506 (Fig. 2). Two-thirds of the surface contact between FKBP12-FK506 and calcineurin B comes from the latch region identified as the major site of interaction of calcineurin B with cyclophilin-CsA (40Milan D. Griffith J. Su M. Roydon Price E. McKeon F. Cell. 1994; 79: 437-447Abstract Full Text PDF PubMed Scopus (99) Google Scholar). This latch region formed by calcineurin B upon binding to calcineurin A may be recognized by each of the two immunosuppressive complexes, explaining their competitive binding to calcineurin (7Liu J. Farmer 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). Thus, the conserved structural features of calcineurin are responsible for the unique ability of calcineurin to interact specifically with two classes of immunosuppressive drugs, CsA and FK506, complexed with their respective binding proteins (as reviewed in Ref. 49Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1968) Google Scholar). The Ca2+ dependence of the phosphatase activity of calcineurin is controlled by two structurally similar but functionally different Ca2+-binding proteins, calmodulin and calcineurin B (5Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar). At less than 10−7m Ca2+, calcineurin B, with its high affinity site occupied, remains bound to calcineurin A, but the enzyme is inactive. Occupancy of the low affinity sites (k d between 0.5 and 1 μm) results in a small activation. The basal activity is stimulated more than 20-fold by the addition of an equimolar amount of calmodulin and is strictly the result of an increasedV max. Consistent with the fact that activation is the result of the Ca2+-dependent binding of calmodulin to calcineurin, the concentration of Ca2+ needed for activation decreases with increasing concentrations of calmodulin, and the calmodulin concentration needed for activation decreases with increasing Ca2+ concentrations (50Stemmer P.M. Klee C.B. Biochemistry. 1994; 33: 6859-6866Crossref PubMed Scopus (244) Google Scholar). The highly cooperative Ca2+ dependence of the calmodulin stimulation of calcineurin (Hill coefficient of 2.5–3) allows the enzyme to respond to narrow Ca2+ thresholds following cell stimulation. As with most calmodulin-regulated enzymes, the mechanism of calmodulin activation is believed to involve binding to the calmodulin-binding domain, resulting in a displacement of an autoinhibitory domain (5Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar,51Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (695) Google Scholar). The flexible structure of the calmodulin-binding domain revealed in the crystal structure of calcineurin and the blocking of the catalytic center by the autoinhibitory domain is compatible with this mechanism, but definitive proof of this mechanism depends on the elucidation of the structure of the calcineurin-calmodulin complex. The displacement of the inhibitory domain upon calmodulin binding can also explain the role of calmodulin in the oxidative inactivation of calcineurin. In crude tissue extracts, calcineurin exhibits a high phosphatase activity that is almost completely dependent on calmodulin and does not depend on added metals for activity but is subject to a time- and Ca2+/calmodulin-dependent inactivation facilitated by small heat-stable inactivators (22Stemmer P.M. Wang X. Krinks M.H. Klee C.B. FEBS Lett. 1995; 374: 237-240Crossref PubMed Scopus (37) Google Scholar). The search for factors responsible for the high phosphatase activity and instability of crude calcineurin led to the finding that, in crude extracts, calcineurin is protected against inactivation by superoxide dismutase (23Wang X. Culotta V.C. Klee C.B. Nature. 1996; 383: 434-437Crossref PubMed Scopus (241) Google Scholar). 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