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• W2022746422 abstract "The human LPIN1 gene encodes the protein lipin 1, which possesses phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase; EC 3.1.3.4) activity (Han, G.-S., Wu, W.-I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210–9218). In this work, we characterized human lipin 1 α, β, and γ isoforms that were expressed in Escherichia coli and purified to near homogeneity. PA phosphatase activities of the α, β, and γ isoforms were dependent on Mg2+ or Mn2+ ions at pH 7.5 at 37 °C. The activities were inhibited by concentrations of Mg2+ and Mn2+ above their optimums and by Ca2+, Zn2+, N-ethylmaleimide, propranolol, and the sphingoid bases sphingosine and sphinganine. The activities were thermally labile at temperatures above 40 °C. The α, β, and γ activities followed saturation kinetics with respect to the molar concentration of PA (Km values of 0.35, 0.24, and 0.11 mm, respectively) but followed positive cooperative (Hill number ∼2) kinetics with respect to the surface concentration of PA (Km values of 4.2, 4.5, and 4.3 mol %, respectively) in Triton X-100/PA-mixed micelles. The turnover numbers (kcat) for the α, β, and γ isoforms were 68.8 ± 3.5, 42.8 ± 2.5, and 5.7 ± 0.2 s−1, respectively, whereas their energy of activation values were 14.2, 15.5, and 18.5 kcal/mol, respectively. The isoform activities were dependent on PA as a substrate and required at least one unsaturated fatty acyl moiety for maximum activity. The human LPIN1 gene encodes the protein lipin 1, which possesses phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase; EC 3.1.3.4) activity (Han, G.-S., Wu, W.-I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210–9218). In this work, we characterized human lipin 1 α, β, and γ isoforms that were expressed in Escherichia coli and purified to near homogeneity. PA phosphatase activities of the α, β, and γ isoforms were dependent on Mg2+ or Mn2+ ions at pH 7.5 at 37 °C. The activities were inhibited by concentrations of Mg2+ and Mn2+ above their optimums and by Ca2+, Zn2+, N-ethylmaleimide, propranolol, and the sphingoid bases sphingosine and sphinganine. The activities were thermally labile at temperatures above 40 °C. The α, β, and γ activities followed saturation kinetics with respect to the molar concentration of PA (Km values of 0.35, 0.24, and 0.11 mm, respectively) but followed positive cooperative (Hill number ∼2) kinetics with respect to the surface concentration of PA (Km values of 4.2, 4.5, and 4.3 mol %, respectively) in Triton X-100/PA-mixed micelles. The turnover numbers (kcat) for the α, β, and γ isoforms were 68.8 ± 3.5, 42.8 ± 2.5, and 5.7 ± 0.2 s−1, respectively, whereas their energy of activation values were 14.2, 15.5, and 18.5 kcal/mol, respectively. The isoform activities were dependent on PA as a substrate and required at least one unsaturated fatty acyl moiety for maximum activity. IntroductionThe PA 2The abbreviations used are: PAphosphatidateDAGdiacylglycerol. phosphatase reaction (see Fig. 1) was first characterized in animal tissues by Kennedy and co-workers in 1957 (1Smith S.W. Weiss S.B. Kennedy E.P. J. Biol. Chem. 1957; 228: 915-922Abstract Full Text PDF PubMed Google Scholar). Subsequent studies during the last quarter of the 20th century implicated PA phosphatase as an important enzyme in lipid metabolism and signaling (2Brindley D.N. Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 3Nanjundan M. Possmayer F. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L1-L23Crossref PubMed Google Scholar, 4Carman G.M. Biochim. Biophys. Acta. 1997; 1348: 45-55Crossref PubMed Scopus (70) Google Scholar, 5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar). The DAG generated in the PA phosphatase reaction may be used by a DAG acyltransferase enzyme to synthesize triacylglycerol and by ethanolaminephosphotransferase or cholinephosphotransferase enzymes to synthesize phosphatidylethanolamine or phosphatidylcholine, respectively (Fig. 1) (2Brindley D.N. Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 3Nanjundan M. Possmayer F. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L1-L23Crossref PubMed Google Scholar, 5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar, 6Carman G.M. Han G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The enzyme substrate PA is also used for phospholipid (e.g. phosphatidylinositol and phosphatidylglycerol) synthesis via CDP-DAG (Fig. 1) (2Brindley D.N. Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 3Nanjundan M. Possmayer F. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L1-L23Crossref PubMed Google Scholar, 6Carman G.M. Han G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). With respect to lipid signaling, PA phosphatase may generate a pool of DAG used for protein kinase C activation, and by the nature of its reaction, it may attenuate the signaling functions of PA (7Exton J.H. J. Biol. Chem. 1990; 265: 1-4Abstract Full Text PDF PubMed Google Scholar, 8Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (916) Google Scholar, 9Testerink C. Munnik T. Trends Plant Sci. 2005; 10: 368-375Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 10Waggoner D.W. Xu J. Singh I. Jasinska R. Zhang Q.X. Brindley D.N. Biochim. Biophys. Acta. 1999; 1439: 299-316Crossref PubMed Scopus (113) Google Scholar, 11Sciorra V.A. Morris A.J. Biochim. Biophys. Acta. 2002; 1582: 45-51Crossref PubMed Scopus (143) Google Scholar). Thus, it is expected that the regulation of PA phosphatase activity not only governs the pathways by which lipids are synthesized but also influences lipid signaling.Studies to establish the roles of PA phosphatase in lipid metabolism and signaling had been hampered by the lack of genetic and molecular information on the enzyme. However, this changed when yeast PAH1 was identified as the gene encoding PA phosphatase, which functions in lipid synthesis, PA signaling, and nuclear/endoplasmic reticulum membrane growth (6Carman G.M. Han G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 12Han G.S. Wu W.I. Carman G.M. J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 13Santos-Rosa H. Leung J. Grimsey N. Peak-Chew S. Siniossoglou S. EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (288) Google Scholar, 14Han G.S. Siniossoglou S. Carman G.M. J. Biol. Chem. 2007; 282: 37026-37035Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 15O'Hara L. Han G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 16Carman G.M. Han G.S. J. Biol. Chem. 2009; 284: 2593-2597Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The homology between the yeast PA phosphatase protein and the lipin proteins encoded by the mouse Lpin1, Lpin2, and Lpin3 genes (17Péterfy M. Phan J. Xu P. Reue K. Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (463) Google Scholar) led to the discovery that mammalian lipins are PA phosphatase enzymes (12Han G.S. Wu W.I. Carman G.M. J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 18Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). That lipin 1 and lipin 2 complement phenotypes exhibited by the yeast pah1Δ mutant (19Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) indicates the evolutionarily conserved functions of the PA phosphatase enzymes. PA phosphatase (previously known as PA phosphatase 1) is specific for PA and catalyzes the Mg2+-dependent PA phosphatase reaction based on a DXDX(T/V) motif within a haloacid dehalogenase-like domain (6Carman G.M. Han G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 12Han G.S. Wu W.I. Carman G.M. J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 14Han G.S. Siniossoglou S. Carman G.M. J. Biol. Chem. 2007; 282: 37026-37035Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The enzyme is distinguished from the family of lipid phosphate phosphatase enzymes (previously known as PA phosphatase 2) that dephosphorylate a broad spectrum of substrates (e.g. PA, lyso-PA, DAG pyrophosphate, sphingoid base phosphates, and isoprenoid phosphates) by a distinct catalytic mechanism that does not require divalent cations (5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar, 6Carman G.M. Han G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 20Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 21Brindley D.N. English D. Pilquil C. Buri K. Ling Z.C. Biochim. Biophys. Acta. 2002; 1582: 33-44Crossref PubMed Scopus (104) Google Scholar). The functions of those lipid phosphate phosphatase enzymes are known to control the signaling properties of lipid phosphates, such as lyso-PA (5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar, 11Sciorra V.A. Morris A.J. Biochim. Biophys. Acta. 2002; 1582: 45-51Crossref PubMed Scopus (143) Google Scholar, 22Smyth S.S. Sciorra V.A. Sigal Y.J. Pamuklar Z. Wang Z. Xu Y. Prestwich G.D. Morris A.J. J. Biol. Chem. 2003; 278: 43214-43223Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 23Tanyi J.L. Morris A.J. Wolf J.K. Fang X. Hasegawa Y. Lapushin R. Auersperg N. Sigal Y.J. Newman R.A. Felix E.A. Atkinson E.N. Mills G.B. Cancer Res. 2003; 63: 1073-1082PubMed Google Scholar).Lpin1, identified by Reue and co-workers (17Péterfy M. Phan J. Xu P. Reue K. Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (463) Google Scholar) through positional cloning, is the gene mutated in the fatty liver dystrophy (fld) mouse (24Langner C.A. Birkenmeier E.H. Ben-Zeev O. Schotz M.C. Sweet H.O. Davisson M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 7994-8003Abstract Full Text PDF PubMed Google Scholar). In their seminal studies, Reue and co-workers (17Péterfy M. Phan J. Xu P. Reue K. Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (463) Google Scholar, 25Phan J. Reue K. Cell Metab. 2005; 1: 73-83Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) showed that the loss of lipin 1 in mice prevents normal adipose tissue development, leading to lipodystrophy and insulin resistance, whereas its overexpression leads to obesity and insulin sensitivity. Thus, the molecular function of lipin 1 as a PA phosphatase enzyme provides a mechanistic basis for why the absence or overexpression of lipin 1 in mice has a major effect on lipid metabolism (12Han G.S. Wu W.I. Carman G.M. J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 17Péterfy M. Phan J. Xu P. Reue K. Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (463) Google Scholar, 25Phan J. Reue K. Cell Metab. 2005; 1: 73-83Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In addition, mice lacking lipin 1 exhibit peripheral neuropathy that is characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity (24Langner C.A. Birkenmeier E.H. Ben-Zeev O. Schotz M.C. Sweet H.O. Davisson M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 7994-8003Abstract Full Text PDF PubMed Google Scholar, 26Langner C.A. Birkenmeier E.H. Roth K.A. Bronson R.T. Gordon J.I. J. Biol. Chem. 1991; 266: 11955-11964Abstract Full Text PDF PubMed Google Scholar, 27Nadra K. de Preux Charles A.S. Médard J.J. Hendriks W.T. Han G.S. Grès S. Carman G.M. Saulnier-Blache J.S. Verheijen M.H. Chrast R. Genes Dev. 2008; 22: 1647-1661Crossref PubMed Scopus (105) Google Scholar). These effects are mediated through the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway that is activated by elevated levels of PA due to the loss of PA phosphatase activity (27Nadra K. de Preux Charles A.S. Médard J.J. Hendriks W.T. Han G.S. Grès S. Carman G.M. Saulnier-Blache J.S. Verheijen M.H. Chrast R. Genes Dev. 2008; 22: 1647-1661Crossref PubMed Scopus (105) Google Scholar). Moreover, the level and compartmentalization of lipin 1 exert a major impact on the assembly and secretion of hepatic very low density lipoprotein (28Bou Khalil M. Sundaram M. Zhang H.Y. Links P.H. Raven J.F. Manmontri B. Sariahmetoglu M. Tran K. Reue K. Brindley D.N. Yao Z. J. Lipid Res. 2009; 50: 47-58Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). With respect to humans, mutations in the LPIN1 gene are associated with metabolic syndrome, type 2 diabetes, and recurrent acute myoglobinuria in children, whereas mutations in the LPIN2 gene are the basis for the anemia and inflammatory disorders associated with the Majeed syndrome (29Reue K. Donkor J. Diabetes. 2007; 56: 2842-2843Crossref PubMed Scopus (12) Google Scholar, 30Reue K. Brindley D.N. J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 31Reue K. Dwyer J.R. J. Lipid Res. 2009; 50: S109-S114Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Recent work has shown that a conserved serine residue that is mutated in lipin 2 of Majeed syndrome patients is essential for PA phosphatase activity (32Donkor J. Zhang P. Wong S. O'Loughlin L. Dewald J. Kok B.P. Brindley D.N. Reue K. J. Biol. Chem. 2009; 284: 29968-29978Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar).In addition to its PA phosphatase activity, lipin 1 functions as a transcriptional coactivator in the regulation of lipid metabolism gene expression (33Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr., J.C. Kelly D.P. Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 34Péterfy M. Phan J. Reue K. J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 35Reue K. Zhang P. FEBS Lett. 2008; 582: 90-96Crossref PubMed Scopus (159) Google Scholar, 36Koh Y.K. Lee M.Y. Kim J.W. Kim M. Moon J.S. Lee Y.J. Ahn Y.H. Kim K.S. J. Biol. Chem. 2008; 283: 34896-34906Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Lipin 1 interacts with a complex containing peroxisome proliferator-activated receptor α (PPARα) and PPARγ coactivator-1α (PGC-1α) to regulate the expression of genes involved in fatty acid oxidation (33Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr., J.C. Kelly D.P. Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). This transcriptional coactivator function is dependent on an LXXIL motif found near the DXDX(T/V) motif but independent of PA phosphatase activity (33Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr., J.C. Kelly D.P. Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar).Lpin1 expression is required for adipocyte differentiation (19Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 34Péterfy M. Phan J. Reue K. J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), and it is stimulated by glucocorticoids (37Manmontri B. Sariahmetoglu M. Donkor J. Bou Khalil M. Sundaram M. Yao Z. Reue K. Lehner R. Brindley D.N. J. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 38Zhang P. O'Loughlin L. Brindley D.N. Reue K. J. Lipid Res. 2008; 49: 1519-1528Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Interestingly, Lpin2 expression is repressed during adipogenesis, indicating that lipin 1 and lipin 2 have distinct and non-redundant functions in adipocytes (19Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In addition, alternative splicing of mouse Lpin1 mRNA gives rise to two lipin 1 isoforms (α and β) that are postulated to play distinct functions in adipogenesis (34Péterfy M. Phan J. Reue K. J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). It is also known that human LPIN1 mRNA expression is induced by sterol depletion (39Ishimoto K. Nakamura H. Tachibana K. Yamasaki D. Ota A. Hirano K. Tanaka T. Hamakubo T. Sakai J. Kodama T. Doi T. J. Biol. Chem. 2009; 284: 22195-22205Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).Lipin 1 is localized to the cytosol, endoplasmic reticulum, and nucleus (30Reue K. Brindley D.N. J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 35Reue K. Zhang P. FEBS Lett. 2008; 582: 90-96Crossref PubMed Scopus (159) Google Scholar, 40Takeuchi K. Reue K. Am. J. Physiol. Endocrinol. Metab. 2009; 296: E1195-E1209Crossref PubMed Scopus (276) Google Scholar). The cellular locations of lipin 1 are influenced by covalent modifications that include phosphorylation (19Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 41Huffman T.A. Mothe-Satney I. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1047-1052Crossref PubMed Scopus (185) Google Scholar, 42Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) and sumoylation (43Liu G.H. Gerace L. PLoS One. 2009; 4: e7031Crossref PubMed Scopus (60) Google Scholar) and by interaction with 14-3-3 proteins (44Péterfy M. Harris T.E. Fujita N. Reue K. J. Biol. Chem. 2010; 285: 3857-3864Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Phosphorylation of lipin 1 is stimulated by insulin and mediated by the mTOR signaling pathway (41Huffman T.A. Mothe-Satney I. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1047-1052Crossref PubMed Scopus (185) Google Scholar). Lipin 1 is also phosphorylated during the mitotic phase of the cell cycle (19Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Phosphorylated forms of lipin 1 are enriched in the cytosolic fraction, whereas the dephosphorylated forms are enriched in the membrane fraction (19Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 42Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The translocation of lipin 1 from the cytosol to the membrane is essential to its function as a PA phosphatase enzyme. The association of lipin 1 with the nucleus, where it functions as a transcriptional coactivator, is facilitated by the sumoylation of the protein (43Liu G.H. Gerace L. PLoS One. 2009; 4: e7031Crossref PubMed Scopus (60) Google Scholar). However, the nuclear localization of lipin 1 is blocked by its interaction with 14-3-3 proteins in the cytosol (44Péterfy M. Harris T.E. Fujita N. Reue K. J. Biol. Chem. 2010; 285: 3857-3864Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).Understanding the biochemical properties of the PA phosphatase activities of the lipin proteins is fundamental to elucidating the mode of action and control of these activities in vivo. Up to now, however, attempts to purify mammalian forms of PA phosphatase for defined biochemical studies have been unsuccessful (3Nanjundan M. Possmayer F. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L1-L23Crossref PubMed Google Scholar, 5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar). In this work, we purified human lipin 1 isoforms (α, β, and γ) and characterized their PA phosphatase activities with respect to their enzymological and kinetic properties. These studies revealed that although the enzymological properties of the lipin 1 isoforms were generally similar, they differed significantly with respect to their kinetic properties. In addition, this work showed that the purified LPIN1-encoded PA phosphatase activities were potently inhibited by sphingoid bases, indicating a connection between human PA phosphatase and sphingolipid metabolism.DISCUSSIONPA phosphatase has emerged as a crucial enzyme in lipid metabolism that has a significant impact on obesity, lipodystrophy, and the metabolic syndrome (29Reue K. Donkor J. Diabetes. 2007; 56: 2842-2843Crossref PubMed Scopus (12) Google Scholar, 30Reue K. Brindley D.N. J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 31Reue K. Dwyer J.R. J. Lipid Res. 2009; 50: S109-S114Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Previous attempts to purify PA phosphatase from mammalian tissues have been unsuccessful because the enzyme is thermally unstable and highly susceptible to proteolytic degradation (5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar, 57Hosaka K. Yamashita S. Numa S. J. Biochem. 1975; 77: 501-509Crossref PubMed Scopus (41) Google Scholar, 58Butterwith S.C. Hopewell R. Brindley D.N. Biochem. J. 1984; 220: 825-933Crossref PubMed Scopus (24) Google Scholar, 59Siess E.A. Hofstetter M.M. Biol. Chem. 2005; 386: 1197-1201Crossref PubMed Scopus (1) Google Scholar). The association of PA phosphatase with other proteins also complicates its purification (59Siess E.A. Hofstetter M.M. Biol. Chem. 2005; 386: 1197-1201Crossref PubMed Scopus (1) Google Scholar). In addition, prior to the studies of Jamal et al. (60Jamal Z. Martin A. Gomez-Muñoz A. Brindley D.N. J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar), lipid phosphate phosphatase enzymes that utilize PA as a substrate had been unknown (5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar, 20Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 42Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), making it difficult to know which phosphatase enzyme was being isolated. Moreover, posttranslational modification may lead to enzyme degradation and/or inactivation and specific interactions with other proteins (15O'Hara L. Han G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 19Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The identification of LPIN1 as the gene encoding PA phosphatase (12Han G.S. Wu W.I. Carman G.M. J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar) allowed us to selectively express, purify, and characterize the human lipin 1 α, β, and γ isoforms.The human lipin 1 isoforms are predicted to have subunit molecular masses of ∼100 kDa, but they were estimated to be ∼115 kDa by SDS-PAGE. Although phosphorylation is known to decrease the electrophoretic mobility of lipin 1 in mammalian cells (41Huffman T.A. Mothe-Satney I. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1047-1052Crossref PubMed Scopus (185) Google Scholar, 42Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), the anomalous electrophoretic behavior of the purified recombinant lipin 1 isoforms was not due to posttranslational modifications. A similar electrophoretic behavior has also been observed for recombinant yeast PAH1-encoded PA phosphatase (12Han G.S. Wu W.I. Carman G.M. J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), and like lipin 1, the electrophoretic mobility of the yeast enzyme decreases further upon its phosphorylation (15O'Hara L. Han G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar).Early studies have provided useful information (e.g. pH optimum, Mg2+ ion requirement, and sensitivity to modifying chemicals like N-ethylmaleimide) about the basic enzymological properties of PA phosphatase from mammalian tissues (2Brindley D.N. Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 5Brindley D.N. Pilquil C. Sariahmetoglu M. Reue K. Biochim. Biophys. Acta. 2009; 1791: 956-961Crossref PubMed Scopus (80) Google Scholar, 18Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 57Hosaka K. Yamashita S. Numa S. J. Biochem. 1975; 77: 501-509Crossref PubMed Scopus (41) Google Scholar, 58Butterwith S.C. Hopewell R. Brindley D.N. Biochem. J. 1984; 220: 825-933Crossref PubMed Scopus (24) Google Scholar, 59Siess E.A. Hofstetter M.M. Biol. Chem. 2005; 386: 1197-1201Crossref PubMed Scopus (1) Google Scholar, 60Jamal Z. Martin A. Gomez-Muñoz A. Brindley D.N. J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar). However, the data derived from this work are difficult to interpret in a definitive way because they have been derived from studies using impure enzymes. The work reported here on the LPIN1-encoded PA phosphatase isoforms did not contain competing and/or modifying enzymes or molecules that inhibit or stimulate activity. Accordingly, the concentration of Mg2+ needed to stimulate the PA phosphatase activities and the concentrations of other effector molecules needed to inhibit the activities were found to be lower than those previously reported. Moreover, for assays with impure enzyme, EDTA is commonly added to inhibit endogenous protease activities, resulting in an increase in the amount of divalent cations needed to affect activity. This work also uncovered the fact that Mn2+ could partially substitute for the Mg2+ dependences of the isoform activities. However, Mg2+ and especially Mn2+ were inhibitory to the PA phosphatase activities at concentrations higher than their optimum levels. In addition, the purified isoforms were potently inhibited by Ca2+ and Zn2+. The inhibition of the activities by Ca2+ has been attributed to its association with the substrate PA to prevent the dephosphorylation reaction (2Brindley D.N. Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 3Nanjundan M. Possmayer F. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L1-L23Crossref PubMed Google Scholar).Previous work on the kinetics of mammalian forms of PA phosphatase has been difficult to interpret because of undefined components in the enzyme preparations. Moreover, the PA phosphatase assays have been performed with PA-containing phospholipid vesicles along with the detergent Tween 20 (2Brindley D.N. Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 18Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 60Jamal Z. Martin A. Gomez-Muñoz A. Brindley D.N. J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar). The availability of the purified LPIN1-encoded isoforms permitted defined kinetic studies on human PA phosphatase using Triton X-100/PA-mixed micelles. Although previous studies have indicated that Triton X-100 inhibits PA phosphatase activity (2Brindley D.N. Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 3Nanjundan M. Possmayer F. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L1-L23Crossref PubMed Google Scholar), the apparent inhibitory effect of the detergent was due to the dilution of PA within the Triton X-100/PA-mixed micelle (55Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Indeed, the LPIN1-encoded isoforms followed surface dilution kinetics (55Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Te" @default.
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