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• W2164078051 abstract "The yeast Pah1p phosphatidate phosphatase, which catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes, is a cytosolic enzyme that associates with the nuclear/endoplasmic reticulum membrane to catalyze the dephosphorylation of phosphatidate to yield diacylglycerol. Pah1p is phosphorylated on seven (Ser-110, Ser-114, Ser-168, Ser-602, Thr-723, Ser-744, and Ser-748) sites that are targets for proline-directed protein kinases. In this work, we showed that the seven sites are phosphorylated by Pho85p-Pho80p, a protein kinase-cyclin complex known to regulate a variety of cellular processes. The phosphorylation of recombinant Pah1p was time- and dose-dependent and dependent on the concentrations of ATP (3.7 μm) and Pah1p (0.25 μm). Phosphorylation reduced (6-fold) the catalytic efficiency (Vmax/Km) of Pah1p and reduced (3-fold) its interaction (Kd) with liposomes. Alanine mutations of the seven sites ablated the inhibitory effect that Pho85p-Pho80p had on Pah1p activity and on the interaction with liposomes. Analysis of pho85Δ mutant cells, phosphate-starved wild type cells, and cells expressing phosphorylation-deficient forms of Pah1p indicated that loss of Pho85p-Pho80p phosphorylation reduced Pah1p abundance. In contrast, lack of Nem1p-Spo7p, the phosphatase complex that dephosphorylates Pah1p at the nuclear/endoplasmic reticulum membrane, stabilized Pah1p abundance. Although loss of phosphorylation caused a decrease in abundance, a greater amount of Pah1p was associated with membranes when compared with phosphorylated enzyme, and the loss of phosphorylation allowed bypass of the Nem1p-Spo7p requirement for Pah1p function in the synthesis of triacylglycerol. The yeast Pah1p phosphatidate phosphatase, which catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes, is a cytosolic enzyme that associates with the nuclear/endoplasmic reticulum membrane to catalyze the dephosphorylation of phosphatidate to yield diacylglycerol. Pah1p is phosphorylated on seven (Ser-110, Ser-114, Ser-168, Ser-602, Thr-723, Ser-744, and Ser-748) sites that are targets for proline-directed protein kinases. In this work, we showed that the seven sites are phosphorylated by Pho85p-Pho80p, a protein kinase-cyclin complex known to regulate a variety of cellular processes. The phosphorylation of recombinant Pah1p was time- and dose-dependent and dependent on the concentrations of ATP (3.7 μm) and Pah1p (0.25 μm). Phosphorylation reduced (6-fold) the catalytic efficiency (Vmax/Km) of Pah1p and reduced (3-fold) its interaction (Kd) with liposomes. Alanine mutations of the seven sites ablated the inhibitory effect that Pho85p-Pho80p had on Pah1p activity and on the interaction with liposomes. Analysis of pho85Δ mutant cells, phosphate-starved wild type cells, and cells expressing phosphorylation-deficient forms of Pah1p indicated that loss of Pho85p-Pho80p phosphorylation reduced Pah1p abundance. In contrast, lack of Nem1p-Spo7p, the phosphatase complex that dephosphorylates Pah1p at the nuclear/endoplasmic reticulum membrane, stabilized Pah1p abundance. Although loss of phosphorylation caused a decrease in abundance, a greater amount of Pah1p was associated with membranes when compared with phosphorylated enzyme, and the loss of phosphorylation allowed bypass of the Nem1p-Spo7p requirement for Pah1p function in the synthesis of triacylglycerol. In the yeast Saccharomyces cerevisiae, the PAH1 gene encodes Pah1p PAP 2The abbreviations used are: PAPphosphatidate phosphatasePAphosphatidatePCphosphatidylcholineDAGdiacylglycerolTAGtriacylglycerolERendoplasmic reticulum3Aalanine mutations of Ser-602, Thr-723, and Ser-7444Aalanine mutations of Ser-110, Ser-114, Ser-168, and Ser-7487Aalanine mutations of Ser-110, Ser-114, Ser-168, Ser-602, Thr-723, Ser-744, and Ser-748PclPho85p cyclin. that has emerged as one of the most important and highly regulated enzymes in lipid metabolism (1Carman G.M. Han G.S. Roles of phosphatidate phosphatase enzymes in lipid metabolism.Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 2Carman G.M. Han G.S. Phosphatidic acid phosphatase, a key enzyme in the regulation of lipid synthesis.J. Biol. Chem. 2009; 284: 2593-2597Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 3Carman G.M. Han G.S. Regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae.Annu. Rev. Biochem. 2011; 80: 859-883Crossref PubMed Scopus (180) Google Scholar). The enzyme catalyzes the dephosphorylation of PA, yielding DAG (4Smith S.W. Weiss S.B. Kennedy E.P. The enzymatic dephosphorylation of phosphatidic acids.J. Biol. Chem. 1957; 228: 915-922Abstract Full Text PDF PubMed Google Scholar) (Fig. 1), a reaction that is dependent on Mg2+ ions and is based on a so-called DXDX(T/V) catalytic motif within a haloacid dehalogenase-like domain in the protein (5Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 6Han G.S. Siniossoglou S. Carman G.M. The cellular functions of the yeast lipin homolog Pah1p are dependent on its phosphatidate phosphatase activity.J. Biol. Chem. 2007; 282: 37026-37035Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The DAG produced by Pah1p PAP activity is used for TAG synthesis and for the synthesis of the major phospholipids phosphatidylethanolamine and PC (2Carman G.M. Han G.S. Phosphatidic acid phosphatase, a key enzyme in the regulation of lipid synthesis.J. Biol. Chem. 2009; 284: 2593-2597Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 3Carman G.M. Han G.S. Regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae.Annu. Rev. Biochem. 2011; 80: 859-883Crossref PubMed Scopus (180) Google Scholar, 7Soto-Cardalda A. Fakas S. Pascual F. Choi H.S. Carman G.M. Phosphatidate phosphatase plays role in zinc-mediated regulation of phospholipid synthesis in yeast.J. Biol. Chem. 2012; 287: 968-977Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In mammalian cells, lipin is the Pah1p ortholog (8Péterfy M. Phan J. Xu P. Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (469) Google Scholar), and its molecular function as a PAP enzyme has been revealed through the discovery that PAH1 encodes PAP in yeast (5Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 9Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). This finding also led to establishing that Pah1p orthologs in humans (5Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 10Han G.S. Carman G.M. Characterization of the human LPIN1-encoded phosphatidate phosphatase isoforms.J. Biol. Chem. 2010; 285: 14628-14638Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), flies (11Valente V. Maia R.M. Vianna M.C. Paçó-Larson M.L. Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions.FEBS J. 2010; 277: 4775-4788Crossref PubMed Scopus (27) Google Scholar, 12Ugrankar R. Liu Y. Provaznik J. Schmitt S. Lehmann M. Lipin is a central regulator of adipose tissue development and function in Drosophila.Mol. Cell. Biol. 2011; 31: 1646-1656Crossref PubMed Scopus (82) Google Scholar), worms (13Golden A. Liu J. Cohen-Fix O. Inactivation of the C. elegans lipin homolog leads to ER disorganization and to defects in the breakdown and reassembly of the nuclear envelope.J. Cell Sci. 2009; 122: 1970-1978Crossref PubMed Scopus (103) Google Scholar), and plants (14Nakamura Y. Koizumi R. Shui G. Shimojima M. Wenk M.R. Ito T. Ohta H. Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 20978-20983Crossref PubMed Scopus (188) Google Scholar, 15Eastmond P.J. Quettier A.L. Kroon J.T. Craddock C. Adams N. Slabas A.R. Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis.Plant Cell. 2010; 22: 2796-2811Crossref PubMed Scopus (145) Google Scholar) are PAP enzymes. phosphatidate phosphatase phosphatidate phosphatidylcholine diacylglycerol triacylglycerol endoplasmic reticulum alanine mutations of Ser-602, Thr-723, and Ser-744 alanine mutations of Ser-110, Ser-114, Ser-168, and Ser-748 alanine mutations of Ser-110, Ser-114, Ser-168, Ser-602, Thr-723, Ser-744, and Ser-748 Pho85p cyclin. The importance of PAP activity to yeast physiology is typified by a variety of mutant phenotypes. Cells with the pah1Δ mutation exhibit elevated levels of PA and free fatty acids and reduced levels of DAG and TAG (5Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 6Han G.S. Siniossoglou S. Carman G.M. The cellular functions of the yeast lipin homolog Pah1p are dependent on its phosphatidate phosphatase activity.J. Biol. Chem. 2007; 282: 37026-37035Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 16Fakas S. Qiu Y. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays a key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The elevated PA content causes the induction of phospholipid synthesis gene expression and the aberrant expansion of the nuclear/ER membrane (5Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 6Han G.S. Siniossoglou S. Carman G.M. The cellular functions of the yeast lipin homolog Pah1p are dependent on its phosphatidate phosphatase activity.J. Biol. Chem. 2007; 282: 37026-37035Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 17Santos-Rosa H. Leung J. Grimsey N. Peak-Chew S. Siniossoglou S. The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (288) Google Scholar). In fact, the total mass of phospholipids in the pah1Δ mutant is about 2-fold greater when compared with that of wild type cells (16Fakas S. Qiu Y. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays a key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The reduced capacity to synthesize DAG leads to defects in vacuole homeostasis and fusion (18Sasser T. Qiu Q.S. Karunakaran S. Padolina M. Reyes A. Flood B. Smith S. Gonzales C. Fratti R.A. The yeast lipin 1 orthologue Pah1p regulates vacuole homeostasis and membrane fusion.J. Biol. Chem. 2012; 287: 2221-2236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and defects in lipid droplet formation (19Adeyo O. Horn P.J. Lee S. Binns D.D. Chandrahas A. Chapman K.D. Goodman J.M. The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets.J. Cell Biol. 2011; 192: 1043-1055Crossref PubMed Scopus (207) Google Scholar). Moreover, the reduced capacity to synthesize TAG renders the pah1Δ mutant acutely sensitivity to fatty acid-induced toxicity (16Fakas S. Qiu Y. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays a key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In addition, loss of Pah1p causes a respiratory deficiency phenotype and sensitivity to growth at elevated temperature (5Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 17Santos-Rosa H. Leung J. Grimsey N. Peak-Chew S. Siniossoglou S. The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (288) Google Scholar). The factors that modulate Pah1p PAP activity include membrane phospholipids (20Wu W.I. Carman G.M. Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by phospholipids.Biochemistry. 1996; 35: 3790-3796Crossref PubMed Scopus (48) Google Scholar), sphingoid bases (21Wu W.I. Lin Y.P. Wang E. Merrill Jr., A.H. Carman G.M. Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by sphingoid bases.J. Biol. Chem. 1993; 268: 13830-13837Abstract Full Text PDF PubMed Google Scholar), nucleotides (22Wu W.I. Carman G.M. Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by nucleotides.J. Biol. Chem. 1994; 269: 29495-29501Abstract Full Text PDF PubMed Google Scholar), and phosphorylation/dephosphorylation (23O'Hara L. Han G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). However, cellular location might be the most important means by which Pah1p activity is regulated. Because Pah1p is primarily associated with the cytosol, its association with the nuclear/ER membrane where the substrate PA resides is essential to Pah1p activity in vivo (5Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Karanasios E. Han G.S. Xu Z. Carman G.M. Siniossoglou S. A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17539-17544Crossref PubMed Scopus (138) Google Scholar). The association of Pah1p with the membrane is primarily regulated by the phosphorylation state of the enzyme (24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Karanasios E. Han G.S. Xu Z. Carman G.M. Siniossoglou S. A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17539-17544Crossref PubMed Scopus (138) Google Scholar): phosphorylated Pah1p is primarily found in the cytosol, whereas dephosphorylated Pah1p is primarily associated with the membrane (24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The phosphorylated enzyme is recruited to the nuclear/ER membrane where it is dephosphorylated by the Nem1p-Spo7p protein phosphatase complex (17Santos-Rosa H. Leung J. Grimsey N. Peak-Chew S. Siniossoglou S. The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (288) Google Scholar, 24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Karanasios E. Han G.S. Xu Z. Carman G.M. Siniossoglou S. A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17539-17544Crossref PubMed Scopus (138) Google Scholar) (see Fig. 1). This dephosphorylation leads to the anchoring of Pah1p to the membrane (via a short N-terminal amphipathic helix), which allows the PAP reaction and TAG synthesis (25Karanasios E. Han G.S. Xu Z. Carman G.M. Siniossoglou S. A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17539-17544Crossref PubMed Scopus (138) Google Scholar). O'Hara et al. (23O'Hara L. Han G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) have identified multiple sites of phosphorylation in Pah1p. Of these sites, seven (Ser-110, Ser-114, Ser-168, Ser-602, Thr-723, Ser-744, and Ser-748) are contained within the minimal (Ser/Thr)-Pro motif that is a target for protein kinases regulated during the cell cycle (23O'Hara L. Han G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Simultaneous mutation of the seven sites to nonphosphorylatable alanine residues renders Pah1p more active and circumvents the requirement of Nem1p-Spo7p for membrane association (23O'Hara L. Han G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Karanasios E. Han G.S. Xu Z. Carman G.M. Siniossoglou S. A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17539-17544Crossref PubMed Scopus (138) Google Scholar). Three of these sites (Ser-602, Thr-723, and Ser-744) are targets of the Cdc28p-cyclin B complex (24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). However, the in vitro phosphorylation of the three sites has essentially no effect on Pah1p PAP activity (24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Moreover, phosphorylation-deficient mutations of these sites have little effect on the physiological function of Pah1p with respect to TAG synthesis (24Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Thus, the phosphorylation of one or more of the remaining sites among the seven is needed to regulate Pah1p function. However, until now, the identity of the relevant kinase(s) responsible for phosphorylating the remaining four sites was unknown. In the present work, we identified Pho85p, together with its cyclin partner Pho80p, as the protein kinase that phosphorylated all seven of the sites within the (Ser/Thr)-Pro motif (Fig. 1). Pho85p is a multifunctional cyclin-dependent protein kinase involved in several signal transduction pathways that affect cell cycle progression and the metabolism of nutrients (26Moffat J. Huang D. Andrews B. Functions of Pho85 cyclin-dependent kinases in budding yeast.Prog. Cell Cycle Res. 2000; 4: 97-106Crossref PubMed Scopus (52) Google Scholar, 27Carroll A.S. O'Shea E.K. Pho85 and signaling environmental conditions.Trends Biochem. Sci. 2002; 27: 87-93Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 28Huang D. Friesen H. Andrews B. Pho85, a multifunctional cyclin-dependent protein kinase in budding yeast.Mol. Microbiol. 2007; 66: 303-314Crossref PubMed Scopus (110) Google Scholar). Herein we show that Pho85p-Pho80p phosphorylation of Pah1p regulates PAP activity, location, abundance, and function in lipid metabolism. All chemicals were reagent grade or better. Difco was the supplier of growth medium supplies. Modifying enzymes, recombinant Vent DNA polymerase, and restriction endonucleases were from New England Biolabs. The DNA gel extraction kit, plasmid DNA purification kit, and nickel-nitrilotriacetic acid-agarose resin were purchased from Qiagen. Aprotinin, benzamidine, bovine serum albumin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, phosphoamino acids, l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, protein A-Sepharose CL-4B, and Triton X-100 were purchased from Sigma-Aldrich. PCR primers were prepared by Genosys Biotechnologies, the QuikChange site-directed mutagenesis kit was from Stratagene, and carrier DNA for yeast transformation was from Clontech. IgG-Sepharose, polyvinylidene difluoride paper, and the enhanced chemifluorescence Western blotting detection kit were from GE Healthcare. DNA size ladders, electrophoresis reagents, immunochemical reagents, molecular mass protein standards, and protein assay reagents were from Bio-Rad. Lipids and thin-layer chromatography plates (cellulose and silica gel 60) were from Avanti Polar Lipids and EM Science, respectively. Radiochemicals were Perkin-Elmer Life Sciences, and scintillation counting supplies and acrylamide solutions were from National Diagnostics. Alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies, alkaline phosphatase-conjugated goat anti-mouse IgG antibodies, mouse anti-phosphoglycerate kinase antibodies, and mouse anti-(phosphoserine/phosphothreonine)-proline (MPM2) antibodies were from Thermo Scientific, Pierce, Invitrogen, and Millipore, respectively. The strains used in this work are listed in Table 1. Escherichia coli strain DH5α was used for the propagation of plasmids. E. coli cells were grown at 37 °C in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7). Ampicillin (100 μg/ml) was added to select for cells carrying plasmids. His6-tagged wild type and phosphorylation-deficient forms of Pah1p were expressed in E. coli BL21(DE3)pLysS cells bearing the indicated PAH1 derivatives of plasmid pET-15b as described by Han et al. (29Han G.S. Sreenivas A. Choi M.G. Chang Y.F. Martin S.S. Baldwin E.P. Carman G.M. Expression of human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A.J. Biol. Chem. 2005; 280: 38328-38336Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Cells were grown to an A600 of 0.5 at room temperature in 500 ml of LB medium containing ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml). Cultures were incubated for 1 h with 1 mm isopropyl β-d-thiogalactoside to induce expression of His6-tagged wild type and mutant forms of Pah1p. Yeast cells were grown in synthetic complete medium. Appropriate amino acids were omitted from the synthetic growth medium to select for cells carrying specific plasmids. High phosphate medium contained 1 mg/ml potassium phosphate, whereas low phosphate medium contained 4.5 μg/ml potassium phosphate plus 1 mg/ml potassium chloride (30Komeili A. O'Shea E.K. Roles of phosphorylation sites in regulating activity of the transcription factor Pho4.Science. 1999; 284: 977-980Crossref PubMed Scopus (248) Google Scholar). The growth regime described by Komeili and O'Shea (30Komeili A. O'Shea E.K. Roles of phosphorylation sites in regulating activity of the transcription factor Pho4.Science. 1999; 284: 977-980Crossref PubMed Scopus (248) Google Scholar) was used to deplete phosphate from cells. Cell numbers in liquid cultures were determined spectrophotometrically at an absorbance of 600 nm. The growth medium was supplemented with agar (2% for yeast or 1.5% for E. coli) for growth on plates.TABLE 1Strains used in this workStrainRelevant characteristicsSource or Ref.E. coliDH5αF− φ80dlacZΔM15Δ (lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rk− mk+) phoA supE44 l−thi-1 gyrA96 relA131Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarBL21(DE3)pLysSF− ompT hsdSB (rB−mB−) gal dcm (DE3)pLysSNovagenBL21(DE3)F− ompT hsdSB (rB−mB−) gal dcm (DE3)InvitrogenS. cerevisiaeRS453MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-5267Han G.S. O'Hara L. Carman G.M. Siniossoglou S. An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth.J. Biol. Chem. 2008; 283: 20433-20442Abstract Full Text Full Text PDF PubMed Scopus (123) Google ScholarSS1026pah1Δ::TRP1 derivative of RS45317Santos-Rosa H. Leung J. Grimsey N. Peak-Chew S. Siniossoglou S. The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (288) Google ScholarSS1132pah1Δ::TRP1 nem1Δ::HIS3 derivative of RS45324Choi H.S. Su W.M. Morgan J.M. Han G.S. Xu Z. Karanasios E. Siniossoglou S. Carman G.M. Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae. Identification of Ser602, Thr723, and Ser744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (91) Google ScholarEY0057K699 ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL+E. O'SheaEY0140K699 ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL + pho85Δ::LEU2E. O'Shea Open table in a new tab Standard protocols were used to isolate genomic and plasmid DNA, digest and ligate DNA, and amplify DNA by PCR (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor L" @default.
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• W2164078051 title "Pho85p-Pho80p Phosphorylation of Yeast Pah1p Phosphatidate Phosphatase Regulates Its Activity, Location, Abundance, and Function in Lipid Metabolism" @default.
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