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Casein Kinase II Phosphorylation of the Yeast Phospholipid Synthesis Transcription Factor Opi1p

2006; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês

10.1074/jbc.m513064200

ISSN

1083-351X

Autores

Yu-Fang Chang, George Carman,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevisiae by repressing the expression of several UASINO-containing genes (e.g. INO1). Opi1p repressor activity is most active in inositol-supplemented cells. Regulation of Opi1p repressor activity is mediated by multiple phosphorylations catalyzed by protein kinases A and C. In this work, we showed that Opi1p was also phosphorylated by casein kinase II. Using purified maltose-binding protein-Opi1p as a substrate, casein kinase II activity was doseand time-dependent and dependent on the concentrations of maltosebinding protein-Opi1p (Km = 25 μg/ml) and ATP (Km = 7 μm). Of three mutations (S10A, S38A, and S239A) in putative phosphorylation sites, 10 only the S10A mutation affected Opi1p phosphorylation. That Ser was a specific target of casein kinase II was confirmed by the loss of a phosphopeptide in the S10A mutant protein. The S10A mutation did not affect phosphorylation of Opi1p by either protein kinase A or protein kinase C. Likewise, phosphorylation of Opi1p by casein kinase II was not affected by mutations in protein kinase A (S31A and S251A) and protein S10A kinase C (S26A) phosphorylation sites. Expression of the OPI1 allele in an opi1Δ mutant attenuated (2-fold) the repressive effect of Opi1p on INO1 expression, and this effect was only observed when cells were grown in the absence of inositol. These data supported the conclusion that casein kinase II phosphorylation at Ser10 played a role in stimulating the repression of INO1 when Opi1p was not in its most active state (i.e. in inositol-deprived cells). The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevisiae by repressing the expression of several UASINO-containing genes (e.g. INO1). Opi1p repressor activity is most active in inositol-supplemented cells. Regulation of Opi1p repressor activity is mediated by multiple phosphorylations catalyzed by protein kinases A and C. In this work, we showed that Opi1p was also phosphorylated by casein kinase II. Using purified maltose-binding protein-Opi1p as a substrate, casein kinase II activity was doseand time-dependent and dependent on the concentrations of maltosebinding protein-Opi1p (Km = 25 μg/ml) and ATP (Km = 7 μm). Of three mutations (S10A, S38A, and S239A) in putative phosphorylation sites, 10 only the S10A mutation affected Opi1p phosphorylation. That Ser was a specific target of casein kinase II was confirmed by the loss of a phosphopeptide in the S10A mutant protein. The S10A mutation did not affect phosphorylation of Opi1p by either protein kinase A or protein kinase C. Likewise, phosphorylation of Opi1p by casein kinase II was not affected by mutations in protein kinase A (S31A and S251A) and protein S10A kinase C (S26A) phosphorylation sites. Expression of the OPI1 allele in an opi1Δ mutant attenuated (2-fold) the repressive effect of Opi1p on INO1 expression, and this effect was only observed when cells were grown in the absence of inositol. These data supported the conclusion that casein kinase II phosphorylation at Ser10 played a role in stimulating the repression of INO1 when Opi1p was not in its most active state (i.e. in inositol-deprived cells). The synthesis of phospholipids in the yeast Saccharomyces cerevisiae is coordinately regulated through genetic and biochemical mechanisms (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 2Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar). Factors that control phospholipid synthesis include the supplementation of inositol, zinc, and carbon source (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 2Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 6Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar, 7Carman G.M. Biochem. Soc. Trans. 2005; 33: 1150-1153Crossref PubMed Google Scholar). Of these nutrients, inositol has been extensively characterized for its role in the regulation of phospholipid synthesis (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 6Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar). Inositol is an essential nutrient for mammalian cells, which is synthesized in yeast via the INO1-encoded inositol-3-phosphate synthase (8Donahue T.F. Henry S.A. J. Biol. Chem. 1981; 256: 7077-7085Abstract Full Text PDF PubMed Google Scholar). It is an essential precursor for the synthesis of phosphatidylinositol and other inositol-containing lipids in yeast and in mammalian cells (6Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar, 9White M.J. Lopes J.M. Henry S.A. Adv. Microbiol. Physiol. 1991; 32: 1-51Crossref PubMed Google Scholar, 10Lester R.L. Dickson R.C. Adv. Lipid Res. 1993; 26: 253-274PubMed Google Scholar, 11Downes C.P. Macphee C.H. Eur. J. Biochem. 1990; 193: 1-18Crossref PubMed Scopus (178) Google Scholar, 12Divecha N. Irvine R.F. Cell. 1995; 80: 269-278Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 13Dove S.K. Cooke F.T. Douglas M.R. Sayers L.G. Parker P.J. Michell R.H. Nature. 1997; 390: 187-192Crossref PubMed Scopus (388) Google Scholar, 14Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (342) Google Scholar, 15Odorizzi G. Babst M. Emr S.D. Trends Biochem. Sci. 2000; 25: 229-235Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). INO1 and genes (e.g. CDS1, CHO1/PSS1, PSD1, CHO2/PEM1, OPI3/PEM2, CKI1, and CPT1) that code for enzymes responsible for the synthesis of phosphatidylcholine are maximally expressed when inositol is absent from the growth medium (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 6Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar). This regulation involves the positive transcription factors Ino2p (16Nikoloff D.M. McGraw P. Henry S.A. Nucleic Acids Res. 1992; 20: 3253Crossref PubMed Scopus (78) Google Scholar) and Ino4p (17Hoshizaki D.K. Hill J.E. Henry S.A. J. Biol. Chem. 1990; 265: 4736-4745Abstract Full Text PDF PubMed Google Scholar) and a UASINO 2The abbreviations used are: UAS, upstream activating sequence; ER, endoplasmic reticulum; PA, phosphatidate; MBP, maltose-binding protein; HA, hemagglutinin; PVDF, polyvinylidene difluoride. 2The abbreviations used are: UAS, upstream activating sequence; ER, endoplasmic reticulum; PA, phosphatidate; MBP, maltose-binding protein; HA, hemagglutinin; PVDF, polyvinylidene difluoride. cis-acting element (5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 18Kodaki T. Nikawa J. Hosaka K. Yamashita S. J. Bacteriol. 1991; 173: 7992-7995Crossref PubMed Google Scholar, 19Lopes J.M. Hirsch J.P. Chorgo P.A. Schulze K.L. Henry S.A. Nucleic Acids Res. 1991; 19: 1687-1693Crossref PubMed Scopus (100) Google Scholar, 20Schuller H.J. Hahn A. Troster F. Schutz A. Schweizer E. EMBO J. 1992; 11: 107-114Crossref PubMed Scopus (102) Google Scholar, 21Schuller H.J. Richter K. Hoffmann B. Ebbert R. Schweizer E. FEBS Lett. 1995; 370: 149-152Crossref PubMed Scopus (55) Google Scholar) in the promoters of the coregulated genes (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 22Ambroziak J. Henry S.A. J. Biol. Chem. 1994; 269: 15344-15349Abstract Full Text PDF PubMed Google Scholar). The UASINO element contains a consensus-binding site (5′-CANNTG-3′) for an Ino2p-Ino4p heterodimer, which is required for maximum expression of the UASINO-containing genes (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 23Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (179) Google Scholar, 24Loewy B.S. Henry S.A. Mol. Cell. Biol. 1984; 4: 2479-2485Crossref PubMed Scopus (56) Google Scholar, 25Schwank S. Ebbert R. Rautenstrauss K. Schweizer E. Schuller H.J. Nucleic Acids Res. 1995; 23: 230-237Crossref PubMed Scopus (109) Google Scholar). Expression of these genes is repressed when inositol is supplemented to the growth medium (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 6Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar). The inositol-mediated repression of these genes requires the ongoing synthesis of phosphatidylcholine (26Gaynor P.M. Gill T. Toutenhoofd S. Summers E.F. McGraw P. Homann M.J. Henry S.A. Carman G.M. Biochim. Biophys. Acta. 1991; 1090: 326-332Crossref PubMed Scopus (36) Google Scholar, 27Morash S.C. McMaster C.R. Hjelmstad R.H. Bell R.M. J. Biol. Chem. 1994; 269: 28769-28776Abstract Full Text PDF PubMed Google Scholar) and is enhanced by the addition of choline to the growth medium (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 6Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar). The negative transcription factor Opi1p is required for the repression of the UASINO-containing genes (28Greenberg M. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar, 29White M.J. Hirsch J.P. Henry S.A. J. Biol. Chem. 1991; 266: 863-872Abstract Full Text PDF PubMed Google Scholar). Based on genetic and biochemical data (3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar), a model for the inositolmediated repression of the UASINO-containing genes has been proposed (30Loewen C.J.R. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (363) Google Scholar). According to the model, Opi1p is associated with the ER through interactions with the integral membrane protein Scs2p (31Loewen C.J. Roy A. Levine T.P. EMBO J. 2003; 22: 2025-2035Crossref PubMed Scopus (436) Google Scholar) and with PA (30Loewen C.J.R. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (363) Google Scholar) when cells are grown without inositol. Upon inositol supplementation, the levels of PA decrease because of its utilization in the synthesis of phosphatidylinositol (30Loewen C.J.R. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (363) Google Scholar, 32Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The decrease in PA results in the loss of Opi1p association with the ER, followed by its translocation into the nucleus (30Loewen C.J.R. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (363) Google Scholar). Opi1p mediates repression of the coregulated phospholipid biosynthetic genes through the UASINO element (33Bachhawat N. Ouyang Q. Henry S.A. J. Biol. Chem. 1995; 270: 25087-25095Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), but not by direct interaction (34Wagner C. Blank M. Strohmann B. Schüller H.J. Yeast. 1999; 15: 843-854Crossref PubMed Scopus (44) Google Scholar). Instead, Opi1p represses transcriptional activation by binding to DNA-bound Ino2p (35Wagner C. Dietz M. Wittmann J. Albrecht A. Schuller H.J. Mol. Microbiol. 2001; 41: 155-166Crossref PubMed Scopus (84) Google Scholar). In addition, the global repressor Sin3p interacts with Opi1p (35Wagner C. Dietz M. Wittmann J. Albrecht A. Schuller H.J. Mol. Microbiol. 2001; 41: 155-166Crossref PubMed Scopus (84) Google Scholar), and this interaction plays a role in Opi1p repressor function (34Wagner C. Blank M. Strohmann B. Schüller H.J. Yeast. 1999; 15: 843-854Crossref PubMed Scopus (44) Google Scholar). Opi1p also functions to control expression of the UASINO-containing genes when cells are grown in the absence of inositol (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar). This conclusion is based on the fact that opi1Δ mutants exhibit elevated expression of the UASINO-containing genes (1Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 3Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (259) Google Scholar, 5Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 6Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar) and excrete inositol (28Greenberg M. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar) because of overexpressed levels of the INO1-encoded inositol-3-phosphate synthase when cells are grown without inositol (8Donahue T.F. Henry S.A. J. Biol. Chem. 1981; 256: 7077-7085Abstract Full Text PDF PubMed Google Scholar, 23Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (179) Google Scholar, 36Klig L.S. Henry S.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3816-3820Crossref PubMed Scopus (60) Google Scholar, 37Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar). The Opi1p transcription factor is phosphorylated on multiple serine residues (38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 39Sreenivas A. Carman G.M. J. Biol. Chem. 2003; 278: 20673-20680Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Protein kinase A (39Sreenivas A. Carman G.M. J. Biol. Chem. 2003; 278: 20673-20680Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and protein kinase C (38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) are involved in this phosphorylation. Ser31 and Ser251 are major phosphorylation sites for protein kinase A (39Sreenivas A. Carman G.M. J. Biol. Chem. 2003; 278: 20673-20680Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), whereas Ser26 is a major protein kinase C phosphorylation site (38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) (see Fig. 1). Phosphorylation of Opi1p at Ser31 and Ser251 mediates the stimulation of the negative regulatory function of Opi1p (39Sreenivas A. Carman G.M. J. Biol. Chem. 2003; 278: 20673-20680Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), whereas phosphorylation at Ser26 attenuates its negative regulatory function (38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The regulation of Opi1p function by phosphorylation via protein kinases A and C occurs in cells grown in the absence or presence of inositol (38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 39Sreenivas A. Carman G.M. J. Biol. Chem. 2003; 278: 20673-20680Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In the present work, we examined the hypothesis that Opi1p was also a target of casein kinase II (protein kinase casein kinase 2) phosphorylation. Casein kinase II is a highly conserved serine/threonine protein kinase that is ubiquitous in eukaryotic organisms and is essential for cell viability in S. cerevisiae (40Glover III, C.V. Prog. Nucleic Acids Res. Mol. Biol. 1998; 59: 95-133Crossref PubMed Scopus (175) Google Scholar, 41Litchfield D.W. Biochem. J. 2003; 369: 1-15Crossref PubMed Scopus (1004) Google Scholar, 42Guerra B. Issinger O.G. Electrophoresis. 1999; 20: 391-408Crossref PubMed Scopus (363) Google Scholar). The enzyme is composed of two catalytic and two regulatory subunits encoded by the CKA1, CKA2, CKB1, and CKB2 genes, respectively (43Chen-Wu J.L. Padmanabha R. Glover C.V. Mol. Cell. Biol. 1988; 8: 4981-4990Crossref PubMed Scopus (92) Google Scholar, 44Reed J.C. Bidwai A.P. Glover C.V.C. J. Biol. Chem. 1994; 269: 18192-18200Abstract Full Text PDF PubMed Google Scholar, 45Bidwai A.P. Reed J.C. Glover C.V. J. Biol. Chem. 1995; 270: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 46Padmanabha R. Glover C.V.C. J. Biol. Chem. 1987; 262: 1829-1835Abstract Full Text PDF PubMed Google Scholar). Of three potential phosphorylation sites (see Fig. 1), Ser10 was identified as a major site. We also showed that phosphorylation of Ser10 played a role in stimulating Opi1p function in cells grown without inositol. This is the first report of the casein kinase II phosphorylation of a membrane phospholipid synthesis transcription factor in yeast. Materials—All of the chemicals were reagent grade. The growth media were obtained from Difco Laboratories. New England Biolabs was the source of MBP, amylose affinity chromatography resin, recombinant Vent DNA polymerase, restriction endonucleases, and modifying enzymes. DNA gel extraction and plasmid DNA purification kits were purchased from Qiagen. The oligonucleotides were synthesized at Genosys Biotechnologies, Inc. Stratagene was the source of the QuikChange site-directed mutagenesis kit. The Yeast Maker yeast transformation system was from Clontech. The radiochemicals were purchased from PerkinElmer Life Sciences. Aprotinin, benzamidine, bovine serum albumin, leupeptin, Nonidet P-40, O-nitrophenyl β-d-galactopyranoside, pepstatin, phenylmethylsulfonyl fluoride, phosphoamino acids, and polyvinylpyrrolidone were purchased from Sigma. Bio-Rad was the source of DNA size ladders, electrophoresis reagents, immunochemical reagents, isopropyl β-d-thiogalactoside, molecular mass protein standards, and protein assay reagents. Mouse monoclonal anti-HA antibodies (12CA5) and goat anti-mouse IgG alkaline phosphatase conjugates were from Roche Applied Science and Pierce, respectively. Anti-phosphoglycerate kinase antibodies were from Molecular Probes. Casein kinase II was purchased from New England BioLabs. Protein kinase A catalytic subunit and protein kinase C were purchased from Promega. Lipids were obtained from Avanti Polar Lipids. Protein A-Sepharose CL-4B beads, Hybond-P PVDF paper, and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham Biosciences. Cellulose thin layer glass plates were from EM Science. Scintillation counting supplies and acrylamide solutions were purchased from National Diagnostics. Strains, Plasmids, and Growth Conditions—The strains and plasmids used in this work are listed in Table 1. Escherichia coli strain DH5α was used for the propagation of plasmids and for the production of MBP-Opi1p fusion proteins. The cells were grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37 °C. Ampicillin (100 μg/ml) was added to cultures of DH5α cells that carried plasmids. For the expression of MBP-Opi1p fusion proteins, cultures (250 ml) were grown to the exponential phase (A600 nm = 0.5) at 37 °C, and the cells were harvested by centrifugation at 5,000 × g for 5 min and resuspended in fresh media containing 0.6 mm isopropyl β-d-thiogalactoside. After incubation for3hat30°C,the induced cells were harvested by centrifugation at 5,000 × g for 5 min, washed with cold 10 mm Tris-HCl buffer (pH 7.4) containing 0.2 m NaCl, 10 mm 2-mercaptoethanol, and 1 mm EDTA, and then stored at –70 °C. The induction was carried out at 30 °C to reduce the degradation of the fusion proteins. The methods for yeast growth were performed as described previously (47Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar, 48Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in complete synthetic medium (49Culbertson M.R. Henry S.A. Genetics. 1975; 80: 23-40Crossref PubMed Google Scholar) containing 2% glucose at 30 °C. For selection of cells bearing plasmids, appropriate amino acids were omitted from the growth medium. Cell numbers in liquid media were determined spectrophotometrically at an absorbance of 600 nm. The media were supplemented with 2% agar for growth on plates.TABLE 1Strains and plasmids used in this workStrain or plasmidGenotype or relevant characteristicsSource or Ref.E. coliDH5αF-, φ80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk- mk+), phoA, supE44, λ-thi-1, gyrA96, relA1Ref. 48Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. 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Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google ScholarpMAL-OPI1S10AOPI1S10A derivative of pMAL-OPI1This workpMAL-OPI1S38AOPI1S38A derivative of pMAL-OPI1This workpMAL-OPI1S239AOPI1S239A derivative of pMAL-OPI1This workpRS415Single-copy E. coli/yeast shuttle vector containing the LEU2 geneRef. 73Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarYEp351Multicopy E. coli/yeast shuttle vector containing the LEU2 geneRef. 74Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1077) Google ScholarpJH354OPI1 gene ligated into the SacI/HindIII site of YEp351Ref. 29White M.J. Hirsch J.P. Henry S.A. J. Biol. Chem. 1991; 266: 863-872Abstract Full Text PDF PubMed Google ScholarpSA1HA sequence inserted into pJH354 after the ATG start codon in the OPI1 geneRef. 38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google ScholarpSA3HA-tagged OPI1 gene from pSA1 ligated into the SacI/HindIII site of pRS415Ref. 38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google ScholarpYC1HA-tagged OPI1S10A derivative of pSA3This workpYC2HA-tagged OPI1S10A from pYC1 ligated into the SacI/HindIII site of YEp351This workpJH359PINO1-lacZ reporter gene construct containing the URA3 geneRef. 19Lopes J.M. Hirsch J.P. Chorgo P.A. Schulze K.L. Henry S.A. Nucleic Acids Res. 1991; 19: 1687-1693Crossref PubMed Scopus (100) Google Scholar Open table in a new tab DNA Manipulations, Amplification of DNA by PCR, and DNA Sequencing—Standard methods were used to prepare genomic and plasmid DNA, to digest DNA with restriction enzymes, and to ligate DNA (48Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformation of E. coli (48Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and yeast (50Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar, 51Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1763) Google Scholar) was performed as described previously. PCRs were optimized according to the methods described by Innis and Gelfand (52Innis M.A. Gelfand D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA1990: 3-12Google Scholar). DNA sequencing reactions were performed by the dideoxy method using Taq polymerase (48Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and analyzed by automated DNA sequencing. Construction of Plasmids—Plasmid pMAL-OPI1 containing the malE-OPI1 fusion gene (38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) was used for the expression of MBP-Opi1p fusion protein. The codons for Ser10, Ser38 and Ser239S10A, in OPI1 were changed to alanine codons. The OPI1, OPI1S38A, and OPI1S239A mutations were constructed by PCR with a QuikChange site-directed mutagenesis kit using appropriate primers and plasmid pMAL-OPI1 as the template. The correct mutations in the OPI1 alleles were confirmed by DNA sequencing. Plasmid pSA3 is a single-copy plasmid that contains the OPI1 gene with the sequence for an HA epitope tag inserted after the start codon (38Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Plasmid pYC1 that bears the HA-OPI1S10A was derive

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