Identification of a Novel Region Critical for Calcineurin Function in Vivo and in Vitro
1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês
10.1074/jbc.274.26.18543
ISSN1083-351X
Autores Tópico(s)Peptidase Inhibition and Analysis
ResumoCalcineurin is a Ca2+/calmodulin-regulated protein phosphatase that plays critical functional roles in T-cell activation and other Ca2+-mediated signal transduction pathways in mammalian cells. In Saccharomyces cerevisiae, calcineurin regulates the transcription of several genes involved in maintaining ion homeostasis (PMC1, PMR1, and PMR2) and cell wall synthesis (FKS2). In this paper, we report the identification and characterization of 11 single amino acid substitutions in the yeast calcineurin catalytic subunit Cna1p. We show that six substitutions (R177G, F211S, S232F, D258V, L259P, and A262P) affect the stability of calcineurin and that two substitutions (V385D and M400R) disrupt the interaction between Cna1p and the calcineurin regulatory subunit Cnb1p. We also identify three mutations (S373P, H375L, and L379S) that are clustered between the catalytic and the calcineurin B subunit-binding domains. These mutations do not significantly affect the ability of Cna1p to interact with Cnb1p, calmodulin, or Fkb1p (FK506-binding protein). However, these residue substitutions dramatically affect calcineurin activity both in vitro and in vivo. Thus, by using a random mutagenesis approach, we have shown for the first time that the linker region of the calcineurin catalytic subunit, as defined by the Ser373, His375, and Leu379residues, is crucial for its function as a phosphatase. Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase that plays critical functional roles in T-cell activation and other Ca2+-mediated signal transduction pathways in mammalian cells. In Saccharomyces cerevisiae, calcineurin regulates the transcription of several genes involved in maintaining ion homeostasis (PMC1, PMR1, and PMR2) and cell wall synthesis (FKS2). In this paper, we report the identification and characterization of 11 single amino acid substitutions in the yeast calcineurin catalytic subunit Cna1p. We show that six substitutions (R177G, F211S, S232F, D258V, L259P, and A262P) affect the stability of calcineurin and that two substitutions (V385D and M400R) disrupt the interaction between Cna1p and the calcineurin regulatory subunit Cnb1p. We also identify three mutations (S373P, H375L, and L379S) that are clustered between the catalytic and the calcineurin B subunit-binding domains. These mutations do not significantly affect the ability of Cna1p to interact with Cnb1p, calmodulin, or Fkb1p (FK506-binding protein). However, these residue substitutions dramatically affect calcineurin activity both in vitro and in vivo. Thus, by using a random mutagenesis approach, we have shown for the first time that the linker region of the calcineurin catalytic subunit, as defined by the Ser373, His375, and Leu379residues, is crucial for its function as a phosphatase. Calcineurin, also known as PP2B, is a Ser/Thr-specific protein phosphatase (1Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). Calcineurin is tightly regulated by Ca2+/calmodulin, and it plays critical functional roles in many calcium-mediated signal transduction pathways. For example, calcineurin is required during T-cell activation for the transcriptional regulation of interleukin 2 (IL2) and other cytokine genes (2Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1490) Google Scholar, 3O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (801) Google Scholar). In human T-cells, calcineurin directly binds to and dephosphorylates the transcription factor NFAT. 1The abbreviations used are: NFAT, nuclear factor of activated T-cells; CnA, calcineurin A subunit; CnB, calcineurin B subunit; CaM, calmodulin; FKBP, FK506-binding protein; CDRE, calcineurin-dependent responsive element; RII, type II regulatory subunit of cAMP-dependent protein kinase; PCR, polymerase chain reaction; WT, wild type.The dephosphorylated NFAT then translocates from the cytosol to the nucleus and activates the transcription of IL2 and other cytokine genes required for T-cell activation and proliferation (4Flanagan W. Corthesy B. Bram R. Crabtree G. Nature. 1991; 352: 803-807Crossref PubMed Scopus (974) Google Scholar, 5Jain 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 (687) Google Scholar). The immunosuppressive drugs FK506 and cyclosporin A, in association with their respective cellular receptor proteins FKBP12 and cyclophilin A, bind to calcineurin and inhibit its phosphatase activity (6Liu J. Farmer J.D. Lane W.L. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3716) Google Scholar), thus preventing the calcineurin-dependent transcriptional activation of cytokine genes and T-cell activation (7Liu J. Immunol. Today. 1993; 14: 290-295Abstract Full Text PDF PubMed Scopus (281) Google Scholar). In the budding yeast Saccharomyces cerevisiae, calcineurin-deficient strains exhibit normal growth under standard conditions (8Liu Y. Ishii S. Tokai M. Tsutsumi H. Ohke O. Akada R. Tanaka K. Tsuchiya E. Fukui S. Miyakawa T. Mol. Gen. Genet. 1991; 227: 52-59Crossref PubMed Scopus (148) Google Scholar, 9Cyert M.S. Kunisawa R. Kaim D. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7376-7380Crossref PubMed Scopus (250) Google Scholar, 10Cyert M.S. Thorner J. Mol. Cell. Biol. 1992; 12: 3460-3469Crossref PubMed Scopus (212) Google Scholar). However, calcineurin function is required for cell viability under some specific growth conditions. For example, mutations that disrupt calcineurin function are lethal in combination with several mutations that impair cell wall synthesis, includingfks1Δ (11Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl A.M. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.M. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (346) Google Scholar, 12Eng W.-K. Faucette L. McLaughlin M.M. Cafferkey R. Koltin Y. Morris R.A. Young P.R. Johnson R.K. Livi G.P. Gene (Amst.). 1994; 151: 61-71Crossref PubMed Scopus (73) Google Scholar, 13Garrett-Engele P. Moilanen B. Cyert M.S. Mol. Cell. Biol. 1995; 15: 4103-4114Crossref PubMed Scopus (165) Google Scholar). FKS1 and FKS2 are a pair of homologous genes responsible for the synthesis of β1,3-glucan (11Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl A.M. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.M. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (346) Google Scholar, 12Eng W.-K. Faucette L. McLaughlin M.M. Cafferkey R. Koltin Y. Morris R.A. Young P.R. Johnson R.K. Livi G.P. Gene (Amst.). 1994; 151: 61-71Crossref PubMed Scopus (73) Google Scholar, 14Mazur P. Morin N. Baginsky W. El-Sherbeini M. Clemas J.A. Nielsen J.B. Foor F. Mol. Cell. Biol. 1995; 15: 5671-5681Crossref PubMed Google Scholar), which is a glucose homopolymer. Since β1,3-glucan is an essential cell wall component, yeast cells require both theFKS1 and FKS2 genes for viability. In afks1Δ mutant, the transcription of FKS2 gene is up-regulated to compensate for the loss of FKS1 function (14Mazur P. Morin N. Baginsky W. El-Sherbeini M. Clemas J.A. Nielsen J.B. Foor F. Mol. Cell. Biol. 1995; 15: 5671-5681Crossref PubMed Google Scholar). Because calcineurin is required for the activation ofFKS2 transcription, strains lacking both FKS1 and calcineurin functions are inviable due to the lack of FKS2expression (11Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl A.M. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.M. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (346) Google Scholar, 12Eng W.-K. Faucette L. McLaughlin M.M. Cafferkey R. Koltin Y. Morris R.A. Young P.R. Johnson R.K. Livi G.P. Gene (Amst.). 1994; 151: 61-71Crossref PubMed Scopus (73) Google Scholar, 13Garrett-Engele P. Moilanen B. Cyert M.S. Mol. Cell. Biol. 1995; 15: 4103-4114Crossref PubMed Scopus (165) Google Scholar). Recently, it has been shown that the calcineurin-dependent FKS2 transcriptional regulation is mediated by a zinc finger containing transcription factorCRZ1/TCN1, which associates with a DNA element (calcineurin-dependent responsive element (CDRE)) located in the promoter of the FKS2 gene (15Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (389) Google Scholar). Calcineurin is also required for the proper regulation of ion homeostasis in yeast; calcineurin-deficient cells fail to grow in the presence of high concentrations of Mn2+, Na+/Li+, OH− ions but are more tolerant to Ca2+ than the wild type cells (16Nakamura T. Liu Y. Hirata D. Namba H. Harada S. Hirokawa T. Miyakawa T. EMBO J. 1993; 12: 4063-4071Crossref PubMed Scopus (232) Google Scholar, 17Mendoza I. Rubio F. Rodriguez-Navarro A. Pardo J.M. J. Biol. Chem. 1994; 269: 8792-8796Abstract Full Text PDF PubMed Google Scholar, 18Farcasanu I.C. Hirata D. Tsuchiya E. Nishiyama F. Miyakawa T. Eur. J. 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The calcineurin holoenzyme consists of the following two subunits: a catalytic A subunit (CnA) and a regulatory B subunit (CnB) (1Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). The two subunits are tightly bound through hydrophobic interactions (22Watanabe Y. Perrino B.A. Chang B.H. Soderling T.R. J. Biol. Chem. 1995; 270: 456-460Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and both CnA and CnB are essential for calcineurin function. The crystal structure of the full-length recombinant human calcineurin and that of a proteolytic fragment of bovine calcineurin (complexed with FKBP12-FK506) have been solved (23Kissinger 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 (707) Google Scholar, 24Griffith 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 (783) Google Scholar). The CnA subunit contains four functional domains as follows: a catalytic domain at the N terminus of the protein, a CnB-binding domain, a CaM-binding domain, and an auto-inhibitory domain at the C terminus (25Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (178) Google Scholar). The catalytic domain shares significant sequence homology with other phosphatases such as PP1, PP2A, and λPP (1Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 26Zhuo S. Clemens J.C. Stone R.L. Dixon J.E. J. Biol. Chem. 1994; 269: 26234-26238Abstract Full Text PDF PubMed Google Scholar, 27Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2242) Google Scholar). Comparisons of the x-ray crystallographic data of calcineurin and PP1 have shown that the active site of calcineurin is extremely well conserved in both its amino acid composition and the three-dimensional structure (23Kissinger 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 (707) Google Scholar, 24Griffith 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 (783) Google Scholar). The active site of calcineurin also contains a binuclear (Fe3+-Zn2+) metal center, and site-specific mutagenesis studies have identified several active site amino acid residues that are critical for in vitro catalysis and/or metal ion binding (28Mertz P. Yu L. Sikkink R. Rusnak F. J. Biol. Chem. 1997; 272: 21296-21302Crossref PubMed Scopus (63) Google Scholar, 29Mondragon A. Griffith E.C. Sun L. Xiong F. Armstrong C. Liu J.O. Biochemistry. 1997; 36: 4934-4942Crossref PubMed Scopus (88) Google Scholar). The CnB-binding domain is composed of an amphipathic α-helix and is located downstream of the catalytic domain (25Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (178) Google Scholar, 30Sikkink R. Haddy A. MacKelvie S. Mertz P. Litwiller R. Rusnak F. Biochemistry. 1995; 34: 8348-8356Crossref PubMed Scopus (40) Google Scholar, 31Perrino B.A. Ng L.Y. Soderling T.R. J. Biol. Chem. 1995; 270: 340-346Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 32Kincaid R.L. Giri P.R. Higuchi S. Tamura J. Dixon S.C. Marietta C.A. Amorese D.A. Martin B.M. J. Biol. Chem. 1990; 265: 11312-11319Abstract Full Text PDF PubMed Google Scholar). The amino acid residues located at the upper part of the α-helix are exclusively non-polar residues. These non-polar residues form a continuous hydrophobic surface to which the CnB subunit binds through hydrophobic interactions (23Kissinger 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 (707) Google Scholar, 24Griffith 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 (783) Google Scholar). Four hydrophobic residues (Val349, Phe350, Phe356, and Val357) located within the CnB-binding domain of human CnA subunit have been shown to be essential for CnA and CnB interactionin vitro (31Perrino B.A. Ng L.Y. Soderling T.R. J. Biol. Chem. 1995; 270: 340-346Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The CaM-binding domain is located downstream of the CnB-binding domain (25Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (178) Google Scholar, 33Kincaid R.L. Martin B.M. Adv. Exp. Med. Biol. 1989; 155: 347-358Crossref Scopus (6) Google Scholar). This part of the protein is highly sensitive to proteolytic digestion. In addition, the CaM-binding domain is invisible in the electron density map of the full-length recombinant human calcineurin (23Kissinger 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 (707) Google Scholar), suggesting that this region is quite flexible (1Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). The auto-inhibitory domain is located at the C terminus of calcineurin, downstream of the CaM-binding domain (25Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (178) Google Scholar, 34Hashimoto Y. Perrino B.A. Soderling T.R. J. Biol. Chem. 1990; 265: 1924-1927Abstract Full Text PDF PubMed Google Scholar). In the absence of calmodulin, the auto-inhibitory domain is situated in close proximity to the active site and inhibits the enzymatic activity (23Kissinger 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 (707) Google Scholar). Ca2+/calmodulin binding to the CaM-binding domain causes conformational changes to displace the auto-inhibitory domain from the active site, thus activating the phosphatase activity (1Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 23Kissinger 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 (707) Google Scholar, 24Griffith 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 (783) Google Scholar). Proteolytic removal of the autoinhibitory domain also activates the enzymatic activity and transforms the truncated calcineurin into a constitutively active, Ca2+/calmodulin-independent protein phosphatase (1Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 25Hubbard M.J. Klee C.B. Biochemistry. 1989; 28: 1868-1874Crossref PubMed Scopus (178) Google Scholar). Calcineurin is a very well conserved protein. The catalytic subunit of yeast calcineurin, which is encoded by two homologous and redundant genes CNA1 and CNA2, share 55% sequence identity with the human CnA subunit (8Liu Y. Ishii S. Tokai M. Tsutsumi H. Ohke O. Akada R. Tanaka K. Tsuchiya E. Fukui S. Miyakawa T. Mol. Gen. Genet. 1991; 227: 52-59Crossref PubMed Scopus (148) Google Scholar, 9Cyert M.S. Kunisawa R. Kaim D. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7376-7380Crossref PubMed Scopus (250) Google Scholar). The yeast regulatory subunit, which is encoded by a single gene CNB1, is 56% identical to the human CnB subunit (10Cyert M.S. Thorner J. Mol. Cell. Biol. 1992; 12: 3460-3469Crossref PubMed Scopus (212) Google Scholar, 35Kuno T. Tanaka H. Mukai J. Chang C. Hiraga K. Miyakawa T. Tanaka C. Biochem. Biophys. Res. Commun. 1991; 180: 1159-1163Crossref PubMed Scopus (86) Google Scholar). To understand better the structure and function of the CnA subunit, especially those concerning the catalytic domain, we conducted an extensive mutational analysis of the yeastCNA1 gene, using a PCR-based random mutagenesis approach (36Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (419) Google Scholar). In this paper, we report the identification of 11 single mutations within the CNA1 gene that cause in vivofunctional defects. We show that six of the mutations (R177G, F211S, S232F, D258V, L259P, and A262P) affect the steady-state levels of the mutant proteins. Two other mutations (V385D and M400R) specifically disrupt the interaction between the CnA and CnB subunits. Finally, we also identify three mutations (S373P, H375L, and L379S) that are clustered within the linker region between the catalytic domain and the CnB-binding domain. These three mutated CnA subunits were capable of binding to CnB and CaM as efficiently as the wild type protein, yet they displayed dramatic functional defects in vivo and biochemical defects in vitro. Thus, our random mutational analysis has defined this (Ser373, His375, Leu379)-containing region as a novel region of the calcineurin catalytic subunit that is critical for its activity. Yeast strain BJY301 (MATa, ura3-52, lys2-801, ade2-101, trp1-Δ63, his3-Δ200, leu2-Δ1, cna1Δ, cna2Δ, ura3-52::4XCDRE-lacZ), a derivative of YPH499 (37Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), was used to generate and study CNA1 mutations. In addition to chromosomal disruptions for both CNA1 and CNA2genes, BJY301 contains a lacZ reporter under the control of four tandem copies of CDRE (calcineurin-dependent responsive element) (15Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (389) Google Scholar). Y190 (MATa, ura3-52, his3-Δ200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4Δ, gal80Δ, URA3::GAL UAS -GAL1 TATA -lacZ, cyh r 2, LYS2::GAL UAS -HIS3 TATA -HIS3) was used for all two-hybrid analyses (38Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4937) Google Scholar, 39Chien C. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1237) Google Scholar). It contains aHIS3 reporter and a lacZ reporter, both of which are under control of a GAL4-responsive element. Yeast cells were grown on standard YPD or SCD media according to Sherman et al. (40Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar), except that amino acid supplements were added in SCD media at twice of the recommended level. Standard procedures were used for genetic manipulation of yeast strains (40Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). Phenotypic characterization of the calcineurin mutant strains was performed as described by Pozos et al. (19Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Crossref PubMed Scopus (129) Google Scholar). The plasmids used in this study are shown in TableI. In some cases, multiple steps were employed for plasmid construction, and detailed procedures for plasmid construction are available upon request. BJP2001 is a pRS314-based plasmid containing the wild type (WT) CNA1 gene (37Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Plasmids BJP2002-2012 are the same as BJP2001, except that each plasmid contains a single mutation, which leads to an amino acid substitution as indicated in parentheses. Plasmids BJP2013-2023 were used for two-hybrid experiments. Plasmid BJP2013 contains the full-lengthCNB1 gene fused in-frame to the GAL4 activation domain. Plasmids BJP2014–2019 contain the full-length CNA1(WT or mutant) gene fused in-frame to the GAL4 DNA-binding domain. Plasmid pHS14 contains the full-length CMD1 gene fused in-frame to the GAL4 DNA-binding domain (41Geiser J.R. Sundberg H.A. Chang B.H. Muller E.G. Davis T.N. Mol. Cell. Biol. 1993; 13: 7913-7924Crossref PubMed Scopus (126) Google Scholar). Plasmids BJP2020–2023 contain the full-length CNA1 (WT or mutant) gene fused in-frame to the GAL4 activation domain. Plasmids BJP2025–2029 are used for overexpression and purification of the wild type or mutant calcineurin protein. BJP2025 is a YEp351 derivative containing the CNB1 gene (42Hill J.E. Myers A.M. Koerner J.J. Tzagaloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1135) Google Scholar). Plasmids BJP2026–2029 contain the CNA1 (WT or mutant) gene under the control of aGAL1-GAL10-inducible promoter.Table IPlasmidsPlasmidDescriptionRef.pRS314YCp vector with the TRP1marker37Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarBJP2001CNA1(WT) in pRS314This studyBJP2002CNA1(R177G) in pRS314This studyBJP2003CNA1(F211S) in pRS314This studyBJP2004CNA1(S232F) in pRS314This studyBJP2005CNA1(D258V) in pRS314This studyBJP2006CNA1(L259P) in pRS314This studyBJP2007CNA1(A262P) in pRS314This studyBJP2008CNA1(S373P) in pRS314This studyBJP2009CNA1(H375L) in pRS314This studyBJP2010CNA1(L379S) in pRS314This studyBJP2011CNA1(V385D) in pRS314This studyBJP2012CNA1(M400R) in pRS314This studyGBT9TRP1 marked two-hybrid vector expressing the GAL4 DNA-binding domain38Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4937) Google Scholar,39Chien C. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1237) Google ScholarGAD2fLEU2 marked two-hybrid vector expressing the GAL4 activation domain38Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4937) Google Scholar,39Chien C. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1237) Google ScholarBJP2013GAD2f-CNB1This studyBJP2014GBT9-CNA1(WT)This studyBJP2015GBT9-CNA1(S373P)This studyBJP2016GBT9-CNA1(H375L)This studyBJP2017GBT9-CNA1(L379S)This studyBJP2018GBT9-CNA1(V385D)This studyBJP2019GBT9-CNA1(M400R)This studyBJP2020GAD2f-CNA1 (WT)This studyBJP2021GAD2f-CNA1(S373P)This studyBJP2022GAD2f-CNA1(H375L)This studyBJP2023GAD2f-CNA1(L379S)This studypHS14GBT9-CMD141Geiser J.R. Sundberg H.A. Chang B.H. Muller E.G. Davis T.N. Mol. Cell. Biol. 1993; 13: 7913-7924Crossref PubMed Scopus (126) Google ScholarBJP2025YEp351-CNB1This studyBJP2026YEp352(gal)-CNA1(WT)This studyBJP2027YEp352(gal)-CNA1(S373P)This studyBJP2028YEp352(gal)-CNA1(H375L)This studyBJP2029YEp352(gal)-CNA1(L379S)This studySee the text for more descriptions. Open table in a new tab See the text for more descriptions. Random mutagenesis was performed by PCR essentially as described previously (36Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (419) Google Scholar), except that we used equal amounts of dATP, dTTP, dGTP, and dCTP in our PCR reactions. In addition, a lower concentration of MnCl2 (40 μm) was utilized to reduce the mutation rate to 1–2 changes per kilobase. The mutagenic PCR products from several independent reaction mixtures were pooled and used directly to transform, together with theSalI-NdeI gapped pRS314-CNA1 plasmid (TRP1 marked), into the yeast strain BJY301 using the lithium acetate method (36Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (419) Google Scholar, 43Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Yeast transformants grown on SCD-Trp plates were replica-plated and incubated at 25 and 37 °C. The β-galactosidase activity of the transformants was determined by colony lift assays using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (15Stathopoulos A.M. Cyert M.S. Genes Dev. 1997; 11: 3432-3444Crossref PubMed Scopus (389) Google Scholar). We screened for transformants that were white (deficient inCDRE-lacZ activation) at 37 °C but blue (capable ofCDRE-lacZ activation) at 25 °C. Plasmids from these colonies were then extracted, amplified in Escherichia coli, and re-transformed into BJY301 for second round colony lift assays. Transformants that passed the second round test were collected, and the plasmids recovered from these colonies were subjected to DNA sequence analysis to identify mutations in the CNA1 gene. When multiple mutations were found within the CNA1 gene, further subcloning procedures were performed to obtain single mutations. We generated the three-dimensional Cna1p structure using the COMPOSER module of SYBYL (Tripos, Inc.). COMPOSER is a knowledge-based homology modeling program. It compares the protein of interest with proteins whose three-dimensional structures are known and uses the x-ray crystallographic coordinates from homologous proteins (with at least 30% sequence identity) to build a three-dimensional model for your protein. In the case of Cna1p modeling, we have used the truncated bovine CnA subunit (which displays 59% identity) as the structural template (24Griffith 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 (783) Google Scholar). The images shown in Fig. 2 are generated by the computer graphic program InsightII (Molecular Simulations Inc.). Procedures for preparation of yeast whole cell lysates, SDS-polyacrylamide gel electrophoresis, and immunodetection of yeast calcineurin catalytic subunits were performed as described previously (44Withee J.L. Sen R. Cyert M.S. Genetics. 1998; 149: 865-878Crossref PubMed Google Scholar). The signals from the Western blot were developed by enhanced chemiluminescence (ECL), scanned, and quantified using the Image/Histogram function of the Adobe Photoshop program (Adobe Systems Inc.). Protein concentrations were determined by the BCA method (Pierce), using bovine serum albumin as protein standards. Yeast cells containing both YEp351-CNB1 and YEp352(gal)-CNA1(WT or mutant) plasmids were grown in 500 ml of SCD-Leu-Ura media at 25 °C. After galactose induction, cells were harvested, resuspended in lysis buffer, and quickly frozen by dropping the cell slurry directly into liquid nitrogen (10Cyert M.S. Thorner J. Mol. Cell. Biol. 1992; 12: 3460-3469Crossref PubMed Scopus (212) Google Scholar). Calcineurin (wild type or mutant) was partially purified by DE52 ion-exchange chromatography and CaM affinity column purification as described previously (45Nakamura T. Tsutsumi H. Mukai H. Kuno T. Miyakawa T. FEBS Lett. 1992; 309: 103-106Crossref PubMed Scopus (20) Google Scholar). Phosphatase activity from these affinity purified calcineurin preparations was measured using the BIOMOL GREEN Calcineurin Assay Kit (BIOMOL Research Laboratories, Inc.). Each reaction (50 μl total volume) contained 50 mm Tris, pH 8.0, 100 mm NaCl, 6 mmMgCl2, 0.5 mm dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.1 mm CaCl2, 1 μmbovine
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