Phosphorylation of Saccharomyces cerevisiae Choline Kinase on Ser30 and Ser85 by Protein Kinase A Regulates Phosphatidylcholine Synthesis by the CDP-choline Pathway
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m205316200
ISSN1083-351X
AutoresYing Yu, Avula Sreenivas, Darin Ostrander, George Carman,
Tópico(s)Fungal and yeast genetics research
ResumoThe Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser30 and Ser85 contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V max/K m = 10.8 μm−1 nmol min−1mg−1) and GPRRASATDV (V max/K m = 0.15 μm−1 nmol min−1mg−1) containing the protein kinase A motif for Ser30 and Ser85, respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser30 to Ala (S30A) and Ser85 to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Δeki1Δ double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of3H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser30 and Ser85 by protein kinase A regulates PC synthesis by the CDP-choline pathway. The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser30 and Ser85 contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V max/K m = 10.8 μm−1 nmol min−1mg−1) and GPRRASATDV (V max/K m = 0.15 μm−1 nmol min−1mg−1) containing the protein kinase A motif for Ser30 and Ser85, respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser30 to Ala (S30A) and Ser85 to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Δeki1Δ double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of3H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser30 and Ser85 by protein kinase A regulates PC synthesis by the CDP-choline pathway. PC 1The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; DAG, diacylglycerol. 1The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; DAG, diacylglycerol. is the most abundant phospholipid in eukaryotic organisms (1Vance D.E. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B.V., Amsterdam1996: 153-181Google Scholar, 2Kent C. Biochim. Biophys. Acta Lipids Lipid Metab. 1997; 1348: 79-90Crossref PubMed Scopus (190) Google Scholar, 3Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). It serves as a major structural component of cellular membranes (1Vance D.E. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B.V., Amsterdam1996: 153-181Google Scholar, 2Kent C. Biochim. Biophys. Acta Lipids Lipid Metab. 1997; 1348: 79-90Crossref PubMed Scopus (190) Google Scholar, 3Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar), pulmonary surfactant (5Rooney S.A. Am. Rev. Respir. Dis. 1985; 131: 439-460PubMed Google Scholar), serum lipoproteins (6Vance J.E. Vance D.E. Can. J. Biochem. Cell Biol. 1985; 63: 870-881Crossref PubMed Scopus (85) Google Scholar), and bile (7Agellon L.B. Walkey C.J. Vance D.E. Kuipers F. Verkade H.J. Hepatology. 1999; 30: 725-729Crossref PubMed Scopus (26) Google Scholar). There is strong interest in understanding the regulation of PC metabolism, because this phospholipid serves as a reservoir for several lipid messengers (e.g. lyso-PC, phosphatidate, DAG, lysophosphatidate, platelet-activating factor, arachidonic acid) (8Kent C. Carman G.M. Trends Biochem. Sci. 1999; 24: 146-150Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). PC is synthesized by two major pathways: the CDP-choline (Kennedy) pathway and the three-step methylation of PE (1Vance D.E. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B.V., Amsterdam1996: 153-181Google Scholar, 2Kent C. Biochim. Biophys. Acta Lipids Lipid Metab. 1997; 1348: 79-90Crossref PubMed Scopus (190) Google Scholar, 3Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar) (Fig. 1). In mammalian cells, PC is primarily synthesized via the CDP-choline pathway, whereas in the yeastSaccharomyces cerevisiae, PE methylation is the primary route of synthesis (8Kent C. Carman G.M. Trends Biochem. Sci. 1999; 24: 146-150Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In S. cerevisiae, PE is derived from PS, which is synthesized from CDP-DAG and serine (i.e.CDP-DAG pathway) (Fig. 1) (3Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). Both pathways play important roles in the growth and metabolism of higher and lower eukaryotic organisms (8Kent C. Carman G.M. Trends Biochem. Sci. 1999; 24: 146-150Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Choline kinase (ATP:choline phosphotransferase; EC 2.7.1.32) is a cytosolic enzyme that catalyzes the committed step in the synthesis of PC by the CDP-choline pathway (9Wittenberg J. Kornberg A. J. Biol. Chem. 1953; 202: 431-444Abstract Full Text PDF PubMed Google Scholar). The enzyme catalyzes the phosphorylation of choline with ATP to form phosphocholine and ADP (Fig. 1) (9Wittenberg J. Kornberg A. J. Biol. Chem. 1953; 202: 431-444Abstract Full Text PDF PubMed Google Scholar). Genes encoding mammalian and yeast forms of choline kinase have been isolated (10Hosaka K. Tanaka S. Nikawa J. Yamashita S. FEBS Lett. 1992; 304: 229-232Crossref PubMed Scopus (40) Google Scholar, 11Uchida T. Yamashita S. J. Biol. Chem. 1992; 267: 10156-10162Abstract Full Text PDF PubMed Google Scholar, 12Uchida T. J. Biochem. (Tokyo). 1994; 116: 508-518Crossref PubMed Scopus (31) Google Scholar, 13Hosaka K. Kodaki T. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google Scholar), and various forms of the enzyme have been purified (14Ishidate K. Nakagomi K. Nakazawa Y. J. Biol. Chem. 1984; 259: 14706-14710Abstract Full Text PDF PubMed Google Scholar, 15Porter T.J. Kent C. J. Biol. Chem. 1990; 265: 414-422Abstract Full Text PDF PubMed Google Scholar, 16Uchida T. Yamashita S. Biochim. Biophys. Acta. 1990; 1043: 281-288Crossref PubMed Scopus (42) Google Scholar, 17Kim K.-H. Voelker D.R. Flocco M.T. Carman G.M. J. Biol. Chem. 1998; 273: 6844-6852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The need to understand the regulation of choline kinase is emphasized by the fact that unregulated levels of this enzyme play a role in the generation of human tumors by ras oncogenes (18Nakagami K. Uchida T. Ohwada S. Koibuchi Y. Morishita Y. Jpn. J. Cancer Res. 1999; 90: 1212-1217Crossref PubMed Scopus (56) Google Scholar, 19Hernandez-Alcoceba R. Fernandez F. Lacal J.C. Cancer Res. 1999; 59: 3112-3118PubMed Google Scholar, 20Nakagami K. Uchida T. Ohwada S. Koibuchi Y. Suda Y. Sekine T. Morishita Y. Jpn. J. Cancer Res. 1999; 90: 419-424Crossref PubMed Scopus (111) Google Scholar, 21Hernandez-Alcoceba R. Saniger L. Campos J. Nunez M.C. Khaless F. Gallo M.A. Espinosa A. Lacal J.C. Oncogene. 1997; 15: 2289-2301Crossref PubMed Scopus (157) Google Scholar). Moreover, methods are being developed where choline kinase activity is used as a marker for cancer (22Hara T. Kosaka N. Kishi H. J. Nucl. Med. 2002; 43: 187-199PubMed Google Scholar, 23DeGrado T.R. Baldwin S.W. Wang S. Orr M.D. Liao R.P. Friedman H.S. Reiman R. Price D.T. Coleman R.E. J. Nucl. Med. 2001; 42: 1805-1814PubMed Google Scholar) and the enzyme is a target for anticancer drug discovery (24Campos J. Nunez M.C. Rodriguez V. Gallo M.A. Espinosa A. Bioorg. Med. Chem. Lett. 2000; 10: 767-770Crossref PubMed Scopus (34) Google Scholar, 25Ramirez D.M. Rodriguez-Gonzalez A. Penalva V. Lucas L. Lacal J.C. Biochem. Biophys. Res. Commun. 2001; 285: 873-879Crossref PubMed Scopus (43) Google Scholar, 26Campos J.M. Nunez M.C. Sanchez R.M. Gomez-Vidal J.A. Rodriguez-Gonzalez A. Banez M. Gallo M.A. Lacal J.C. Espinosa A. Bioorg. Med. Chem. 2002; 10: 2215-2231Crossref PubMed Scopus (39) Google Scholar). Because of its tractable genetics and ease of molecular manipulation,S. cerevisiae serves as an excellent eukaryotic model to study the regulation of choline kinase. The enzyme is encoded by theCKI1 gene (13Hosaka K. Kodaki T. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google Scholar). Its deduced protein product contains a conserved phosphotransferase consensus sequence (27Brenner S. Nature. 1987; 329: 21Crossref PubMed Scopus (104) Google Scholar) (Fig. 2) believed to be involved in catalytic function (28Yamashita S. Hosaka K. Biochim. Biophys. Acta. 1997; 1348: 63-69Crossref PubMed Scopus (22) Google Scholar, 29Ishidate K. Biochim. Biophys. Acta. 1997; 1348: 70-78Crossref PubMed Scopus (63) Google Scholar). The CKI1 gene is not essential in S. cerevisiae (13Hosaka K. Kodaki T. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google Scholar) because PC is also synthesized by PE methylation (3Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 30Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar). Nevertheless, choline kinase and the CDP-choline pathway become essential for PC synthesis when enzymes in the CDP-DAG pathway are defective (3Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 30Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar). Indeed, mutants defective in the synthesis of PS, PE, or PC are choline auxotrophs (3Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 30Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar). The expression of choline kinase is regulated by growth phase and by supplementation with water-soluble phospholipid precursors (28Yamashita S. Hosaka K. Biochim. Biophys. Acta. 1997; 1348: 63-69Crossref PubMed Scopus (22) Google Scholar). Choline kinase mRNA and protein levels are highest in exponential phase and decline in the stationary phase (28Yamashita S. Hosaka K. Biochim. Biophys. Acta. 1997; 1348: 63-69Crossref PubMed Scopus (22) Google Scholar). Similar to other phospholipid synthetic enzymes (4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar), choline kinase is repressed by the addition of inositol and choline to the growth medium (28Yamashita S. Hosaka K. Biochim. Biophys. Acta. 1997; 1348: 63-69Crossref PubMed Scopus (22) Google Scholar). Yeast choline kinase is also regulated by biochemical mechanisms. Studies with purified enzyme have shown that its substrate ATP and its product ADP allosterically regulate activity (17Kim K.-H. Voelker D.R. Flocco M.T. Carman G.M. J. Biol. Chem. 1998; 273: 6844-6852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). ATP regulates the enzyme by promoting the oligomerization of the enzyme. ADP inhibits choline kinase activity by a mechanism that affects the catalytic properties of the enzyme and the apparent affinity the enzyme has for the substrates ATP and choline (17Kim K.-H. Voelker D.R. Flocco M.T. Carman G.M. J. Biol. Chem. 1998; 273: 6844-6852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Phosphorylation is another mechanism by which yeast choline kinase is regulated (31Kim K.-H. Carman G.M. J. Biol. Chem. 1999; 274: 9531-9538Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The enzyme is phosphorylated on multiple serine residues in vivo, and some of this phosphorylation is mediated by protein kinase A via the Ras-cAMP pathway (31Kim K.-H. Carman G.M. J. Biol. Chem. 1999; 274: 9531-9538Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In vitro, protein kinase A phosphorylates pure choline kinase on a serine residue, and this phosphorylation results in a stimulation of choline kinase activity by a mechanism that increases catalytic turnover (31Kim K.-H. Carman G.M. J. Biol. Chem. 1999; 274: 9531-9538Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The consequence of this phosphorylation on PC synthesis is unknown and is the subject of this paper. Herein, we report the identification of Ser30and Ser85 as target sites of protein kinase A phosphorylation and show that cells bearing S30A and S85A mutations in choline kinase exhibited defects in PC synthesis via the CDP-choline pathway. All chemicals were reagent grade. Growth medium supplies were from Difco. Restriction enzymes, modifying enzymes, and vent DNA polymerase were from New England Biolabs. Polymerase chain reaction and sequencing primers were prepared commercially by Genosys Biotechnologies, Inc. The QuikChange site-directed mutagenesis kit was purchased from Stratagene. The Prism DyeDeoxy DNA sequencing kit was from Applied Biosystems. The Yeastmaker yeast transformation system was from CLONTECH. The DNA size ladder used for agarose gel electrophoresis was from Invitrogen. The plasmid DNA purification and DNA gel extraction kits were from Qiagen, Inc. Phenylmethylsulfonyl fluoride, bovine serum albumin, histone, benzamidine, aprotinin, leupeptin, pepstatin, polyvinylpyrrolidone, standard phosphoamino acids, choline, phosphocholine, and CDP-choline were purchased from Sigma. The protein kinase A catalytic subunit (bovine heart) was purchased from Promega. Phospholipids were purchased from Avanti Polar Lipids. Silica Gel 60 thin layer chromatography plates and cellulose thin layer glass plates were from EM Science. DE52 (DEAE-cellulose) was from Whatman. Radiochemicals were purchased from PerkinElmer Life Sciences. Phosphocellulose filters were purchased from Pierce. Protein assay reagents, electrophoretic reagents, and immunochemical reagents were purchased from Bio-Rad. Protein A-Sepharose CL-4B beads, polyvinylidene difluoride membrane, and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham Biosciences. Scintillation counting supplies and acrylamide solutions were purchased from National Diagnostics. Peptides were synthesized and purified commercially by Bio-Synthesis, Inc. The strains and plasmids used in this work are listed in TableI. Methods for growth and analysis of yeast were performed as described previously (32Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar, 33Sambrook 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 complete synthetic medium minus inositol (34Culbertson M.R. Henry S.A. Genetics. 1975; 80: 23-40Crossref PubMed Google Scholar), containing 2% glucose and 100 μm choline at 30 °C. Cells were incubated with 100 μm[methyl-3H]choline (0.3 μCi/ml) and with32Pi (5 μCi/ml) for five to six generations to label CDP-choline pathway intermediates and phospholipids. Forin vivo labeling of phosphorylated choline kinase, exponential phase cells were incubated with32Pi (250 μCi/ml) for 3 h. Plasmid maintenance and amplifications were performed in Escherichia coli strain DH5α. E. coli 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 cells that carried plasmids. For growth on plates, the media were supplemented with either 2% (yeast) or 1.5% (E. coli) agar. Yeast cell numbers in liquid media were determined spectrophotometrically at an absorbance of 600 nm. The choline excretion phenotype (35Patton-Vogt J.L. Griac P. Sreenivas A. Bruno V. Dowd S. Swede M.J. Henry S.A. J. Biol. Chem. 1997; 272: 20873-20883Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) was examined on complete synthetic medium plates (minus inositol and choline) by using growth of a choline auxotrophic cho2 opi3 (pem1 pem2) double mutant (36Kodaki T. Yamashita S. J. Biol. Chem. 1987; 262: 15428-15435Abstract Full Text PDF PubMed Google Scholar, 37Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar).Table IStrains and plasmids used in this workStrain or plasmidGenotype or relevant characteristicsSource or referenceE. coliDH5αF−φ80dlacZΔM15 Δ(lacZYA-argF)U169deoR recA1 endA1 hsdR17 (rk − mk +)phoA supE44 λ− thi-1 gyrA96relA1Ref. 33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarS. cerevisiaeKS106MATαleu2–3 112, trp1–1 can1–100 ura3–1 ade2–1 his3–11,15 ekilΔ::TRP1 cki1 Δ::HIS3Ref. 59Kim K. Kim K.-H. Storey M.K. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 274: 14857-14866Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarKS119MATα lys2–801 trp1 ura3–52 his3–200 sec14–3tscki1Δ::HIS3Ref. 59Kim K. Kim K.-H. Storey M.K. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 274: 14857-14866Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarSH920MATα his3 trp1 ade2 leu2 ura3 cho2::LEU2 opi3::URA3S. A. HenryPlasmidspCK1DMultipcopy plasmid containing theCKI1 geneRef. 13Hosaka K. Kodaki T. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google ScholarpBlueScript IIColor-selectable multiple cloning site plasmidStratagenepDO227pBlueScript II containing a 2.7-kbHindIII/PstI fragment of pCK1DThis workYEp351Multicopy E. coli/yeast shuttle vector containing LEU2Ref. 78Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1080) Google ScholarpYY264CKI1derivative of YEp351This workpYY265CKI1 S30A derivative of YEp351This workpYY266CKI1 S85Aderivative of YEp351This workpYY267CKI1 S30A,S85A derivative of YEp351This workpRS416Cen-based single-copy E. coli/yeast shuttle vector containing URA3Ref. 79Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarpYY274CKI1 derivative of pRS416This workpYY275CKI1 S30A derivative of pRS416This workpYY276CKI1 S85Aderivative of pRS416This workpYY277CKI1 S30A,S85A derivative of pRS416This work Open table in a new tab Genomic and plasmid DNA preparation, digestion with restriction enzymes, and DNA ligations were performed as described previously (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformation of yeast (38Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar, 39Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1773) Google Scholar) and E. coli (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) were performed by standard methods. Conditions for DNA amplification by PCR were optimized as described by Innis and Gelfand (40Innis 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 Diego1990: 3-12Google Scholar). DNA sequencing reactions were performed by the dideoxy method using Taq polymerase (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and analyzed with an automated DNA sequencer. TheCKI1 S30A and CKI1 S85Awere constructed by PCR with the QuikChange site-directed mutagenesis kit using plasmid pDO227 as the template. Plasmid pDO227 was constructed by subcloning the 2.7-kb HindIII/PstI fragment of plasmid pCK1D (13Hosaka K. Kodaki T. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google Scholar) into pBlueScript II. The oligonucleotides for the S30A (5′-GAGTTCTCAAAGAAGgCATgCGTTAACACGCCAAC-3′) and S85A (5′-GGGACCAAGAAGAGCtgCAGCAACTGATGTCA-3′) mutations and their complements incorporated SphI and PstI restriction sites, respectively. These silent restriction sites were used to identify plasmids with the correct mutation. A third mutagenesis reaction was performed to combine both the S30A and S85A mutations in a single allele. The mutated genes were completely sequenced to verify that no additional mutations were made. The wild-type and S30A, S85A, and S30A,S85A mutant alleles ofCKI1 were released from pDO227 by digestion withHindIII/XbaI. The resulting 2.7-kb fragments of the wild-type and mutant alleles were ligated into plasmid YEp351 to form the multicopy shuttle vectors pYY264-pYY267 and into plasmid pRS416 to form the single copy shuttle vectors pYY274-pYY277 (Table I). Plasmids YEp351 and pRS416 were digested withHindIII/XbaI before the ligations. Theeki1Δ cki1Δ double mutant strain KS106 was transformed to leucine prototrophy with the multicopy plasmids containing the wild-type and S30A, S85A, and S30A,S85A mutant alleles of CKI1. The sec14 ts cki1Δ double mutant strain KS119 was transformed to uracil prototrophy with the single copy plasmids containing the wild-type and mutant alleles of CKI1. The peptide sequence VQESRPGSVRSYSVGYQ (residues 2–18 at the N-terminal end of the deduced protein sequence of CKI1) was synthesized and conjugated to carrier protein at Bio-Synthesis, Inc. (Lewisville, TX). Antibodies were raised against the choline kinase peptide in New Zealand White rabbits by standard procedures (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) at Bio-Synthesis, Inc. The specificity of the anti-choline kinase peptide antibodies was examined systematically by performing immunoprecipitation and immunoblotting experiments using various concentrations of antiserum and pure choline kinase. For immunoprecipitation experiments, cells were disrupted with glass beads in radioimmune immunoprecipitation buffer (50 mmTris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) containing protease (0.5 mm phenylmethylsulfonyl fluoride, 1 mmbenzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin) and phosphatase (10 mm NaF, 5 mm β-glycerophosphate, and 1 mm sodium vanadate) inhibitors. 0.5 ml of cell extract (1 mg/ml protein) was precleared by incubation with 0.15 ml of protein A-Sepharose CL-4B beads (10% suspension, w/v) for 1 h at 4 °C. Following incubation, the beads were removed by centrifugation at 1,000 × g for 30 s. Choline kinase was immunoprecipitated from the cleared supernatant by incubation with 5 μl of anti-choline kinase peptide antiserum for 1.5 h followed by incubation with 0.15 ml of protein A-Sepharose CL-4B beads for 1 h. The beads were collected by centrifugation at 1,000 ×g for 30 s and washed three times with 50 mm Tris-HCl buffer (pH 8.0) containing 150 mmNaCl and 10 mm MgCl2. Following the washing steps, the buffer was removed by aspiration, and the choline kinase attached to the protein A-Sepharose CL-4B beads was used as substrate for protein kinase A phosphorylation. For in vivo labeling experiments, cell extracts were prepared with 50 mmTris-HCl (pH 8.0) buffer containing protease and phosphatase inhibitors. Following immunoprecipitation, choline kinase proteins were dissociated from enzyme-antibody complexes (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar), subjected to SDS-polyacrylamide gel electrophoresis (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar), and transferred to polyvinylidene difluoride membranes (43Haid A. Suissa M. Methods Enzymol. 1983; 96: 192-205Crossref PubMed Scopus (232) Google Scholar). The 32P-labeled proteins were visualized and quantified by PhosphorImaging analysis. For immunoblotting experiments, protein samples on polyvinylidene difluoride membranes were probed with a 1:5000 dilution of anti-choline kinase peptide antibodies. The choline kinase protein was detected using the ECF Western blotting chemifluorescent detection kit as described by the manufacturer. The choline kinase protein on immunoblots was acquired by FluorImaging analysis. The relative density of the protein was analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability. Immunoprecipitated choline kinase and choline kinase synthetic peptides were phosphorylated with protein kinase A using the bovine heart catalytic subunit. This enzyme is structurally and functionally similar to the S. cerevisiae protein kinase A catalytic subunit (44Toda T. Cameron S. Sass P. Zoller M. Wigler M. Cell. 1987; 50: 277-287Abstract Full Text PDF PubMed Scopus (506) Google Scholar) and phosphorylates pure choline kinase under zero order kinetics (31Kim K.-H. Carman G.M. J. Biol. Chem. 1999; 274: 9531-9538Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Phosphorylation reactions were measured for 10 min at 30 °C in a total volume of 40 μl. Reaction mixtures contained 50 mm Tris-HCl (pH 7.5), 60 mmdithiothreitol, 15 μm [γ-32P]ATP (4 μCi/nmol), 10 mm MgCl2, protein kinase A, and immunoprecipitated choline kinase or synthetic peptides. For samples containing the immunoprecipitated choline kinase, the reaction was terminated by the addition of 1 ml of ice-cold radioimmune immunoprecipitation buffer. The protein A-Sepharose CL-4B beads were collected by centrifugation and washed three times with the same buffer. The beads were suspended in Laemmli sample buffer and subjected to SDS-polyacrylamide gel electrophoresis (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar) followed by transfer to polyvinylidene difluoride membranes (43Haid A. Suissa M. Method
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