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• W2763669665 abstract "Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro. The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro. Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro. The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro. In vertebrates, the lipin family of Mg2+-dependent phosphatidic acid (PA) 3The abbreviations used are: PAphosphatidic acidmTORmammalian target of rapamycinPEphosphatidylethanolaminePBDpolybasic domainNEMN-ethylmaleimideβMEβ-mercaptoethanolSRDserine-rich domainPCphosphatidylcholine. phosphatases consists of three members (lipins 1–3), which form diacylglycerol from PA in neutral and phospholipid synthesis (1Coleman R.A. Lee D.P. Enzymes of triacylglycerol synthesis and their regulation.Prog. Lipid Res. 2004; 43: 134-176Crossref PubMed Scopus (708) Google Scholar2Carman 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. Roles of phosphatidate phosphatase enzymes in lipid metabolism.Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 4Reue K. Zhang P. The lipin protein family: dual roles in lipid biosynthesis and gene expression.FEBS Lett. 2008; 582: 90-96Crossref PubMed Scopus (161) Google Scholar, 5Harris T.E. Finck B.N. Dual function lipin proteins and glycerolipid metabolism.Trends Endocrinol. Metab. 2011; 22: 226-233Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar6Reue K. Dwyer J.R. Lipin proteins and metabolic homeostasis.J. Lipid Res. 2009; 50: S109-S114Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Lipins are cytosolic enzymes that must associate with membranes to access substrate (1Coleman R.A. Lee D.P. Enzymes of triacylglycerol synthesis and their regulation.Prog. Lipid Res. 2004; 43: 134-176Crossref PubMed Scopus (708) Google Scholar, 7Brindley D.N. Intracellular translocation of phosphatidate phosphohydrolase and its possible role in the control of glycerolipid synthesis.Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 8Gomez-Muñoz A. Hatch G.M. Martin A. Jamal Z. Vance D.E. Brindley D.N. Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes.FEBS Lett. 1992; 301: 103-106Crossref PubMed Scopus (44) Google Scholar9Grimsey N. Han G.S. O'Hara L. Rochford J.J. Carman G.M. Siniossoglou S. Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2.J. Biol. Chem. 2008; 283: 29166-29174Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Their spatial regulation has been studied most extensively in the context of insulin signaling (10Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar11Huffman T.A. Mothe-Satney I. Lawrence Jr., J.C. Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1047-1052Crossref PubMed Scopus (188) Google Scholar, 12Peterson T.R. Sengupta S.S. Harris T.E. Carmack A.E. Kang S.A. Balderas E. Guertin D.A. Madden K.L. Carpenter A.E. Finck B.N. Sabatini D.M. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway.Cell. 2011; 146: 408-420Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar13Péterfy M. Harris T.E. Fujita N. Reue K. Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes.J. Biol. Chem. 2010; 285: 3857-3864Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). More specifically, inhibition of mTOR and reduction in the phosphorylation of lipin 1 allow its translocation to the endoplasmic reticulum membrane and nucleus (10Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar11Huffman T.A. Mothe-Satney I. Lawrence Jr., J.C. Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1047-1052Crossref PubMed Scopus (188) Google Scholar, 12Peterson T.R. Sengupta S.S. Harris T.E. Carmack A.E. Kang S.A. Balderas E. Guertin D.A. Madden K.L. Carpenter A.E. Finck B.N. Sabatini D.M. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway.Cell. 2011; 146: 408-420Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar, 13Péterfy M. Harris T.E. Fujita N. Reue K. Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes.J. Biol. Chem. 2010; 285: 3857-3864Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar14Eaton J.M. Mullins G.R. Brindley D.N. Harris T.E. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.J. Biol. Chem. 2013; 288: 9933-9945Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We have shown that whereas lipin 1 is regulated by phosphorylation in vitro, the activity and localization of lipin 2 are not (15Eaton J.M. Takkellapati S. Lawrence R.T. McQueeney K.E. Boroda S. Mullins G.R. Sherwood S.G. Finck B.N. Villén J. Harris T.E. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.J. Biol. Chem. 2014; 289: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). phosphatidic acid mammalian target of rapamycin phosphatidylethanolamine polybasic domain N-ethylmaleimide β-mercaptoethanol serine-rich domain phosphatidylcholine. Lipins are PA effector proteins and are regulated by the lipid’s intrinsic chemical properties (16Stace C.L. Ktistakis N.T. Phosphatidic acid- and phosphatidylserine-binding proteins.Biochim. Biophys. Acta. 2006; 1761: 913-926Crossref PubMed Scopus (300) Google Scholar, 17Shin J.J. Loewen C.J. Putting the pH into phosphatidic acid signaling.BMC Biol. 2011; 9: 85Crossref PubMed Scopus (89) Google Scholar). The PA headgroup is a negatively charged phosphomonoester and contains two dissociable protons with pKa values of 3 and ∼7.9, pKa1 and pKa2, respectively (18Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special?.Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (133) Google Scholar, 19Kooijman E.E. Burger K.N. Biophysics and function of phosphatidic acid: a molecular perspective.Biochim. Biophys. Acta. 2009; 1791: 881-888Crossref PubMed Scopus (114) Google Scholar). At physiological pH, PA always carries a charge of at least −1. However, at least two phenomena can cause PA to become di-anionic: proximity to a hydrogen bond donor, which lowers the pKa2 below the physiological pH, or increase in the membrane pH above that of pKa2 (19Kooijman E.E. Burger K.N. Biophysics and function of phosphatidic acid: a molecular perspective.Biochim. Biophys. Acta. 2009; 1791: 881-888Crossref PubMed Scopus (114) Google Scholar). Positively charged, basic amino acids and phosphatidylethanolamine (PE), an abundant membrane lipid, are sources of hydrogen bond donors (18Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special?.Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (133) Google Scholar, 20van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4480) Google Scholar). Although there is no canonical lipid-binding site on PA effector proteins, many of them contain a cluster of lysines and/or arginines critical for binding to PA (16Stace C.L. Ktistakis N.T. Phosphatidic acid- and phosphatidylserine-binding proteins.Biochim. Biophys. Acta. 2006; 1761: 913-926Crossref PubMed Scopus (300) Google Scholar). Kooijman (21Kooijman E.E. Tieleman D.P. Testerink C. Munnik T. Rijkers D.T. Burger K.N. de Kruijff B. An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins.J. Biol. Chem. 2007; 282: 11356-11364Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) et al. postulated that these residues are responsible for the initial attraction of PA effectors to the negatively charged membrane. The proteins sample the surface until they encounter a mono-anionic PA. They hydrogen-bond with the PA headgroup and cause a dissociation of the final proton. This causes a switch in the charge from mono- to di-anionic and locks the protein onto its lipid-ligand through strengthened electrostatic interactions. This model was termed the hydrogen bond switch mechanism and has been demonstrated experimentally by an induction in lipin activity in the presence of di-anionic PA (14Eaton J.M. Mullins G.R. Brindley D.N. Harris T.E. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.J. Biol. Chem. 2013; 288: 9933-9945Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Eaton J.M. Takkellapati S. Lawrence R.T. McQueeney K.E. Boroda S. Mullins G.R. Sherwood S.G. Finck B.N. Villén J. Harris T.E. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.J. Biol. Chem. 2014; 289: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 21Kooijman E.E. Tieleman D.P. Testerink C. Munnik T. Rijkers D.T. Burger K.N. de Kruijff B. An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins.J. Biol. Chem. 2007; 282: 11356-11364Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Each lipin contains a cluster of nine lysines and arginines known as the polybasic domain (PBD). This region on lipin 1 was identified as the primary PA-binding site and a membrane/nuclear localization sequence (13Péterfy M. Harris T.E. Fujita N. Reue K. Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes.J. Biol. Chem. 2010; 285: 3857-3864Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 22Ren H. Federico L. Huang H. Sunkara M. Drennan T. Frohman M.A. Smyth S.S. Morris A.J. A phosphatidic acid binding/nuclear localization motif determines lipin1 function in lipid metabolism and adipogenesis.Mol. Biol. Cell. 2010; 21: 3171-3181Crossref PubMed Scopus (54) Google Scholar, 23Péterfy 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 (468) Google Scholar). In addition, it is likely to also be the site of negative regulation of lipin 1 by phosphorylation (14Eaton J.M. Mullins G.R. Brindley D.N. Harris T.E. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.J. Biol. Chem. 2013; 288: 9933-9945Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The PBD is conserved among PAP enzymes, but the specific arrangement and number of lysines and arginines vary between the mammalian lipins 1–3. The present study is the first to investigate the in vitro activity of purified lipin 3 against PA-containing liposomes in the context of phosphorylation and the electrostatic charge of PA. Additionally, we present evidence that the specific PBDs of lipins 1 and 3 are responsible for the observed differences in their in vitro phosphoregulation. Adenovirus was used to overexpress Mus musculus FLAG-lipin 3 in HeLa cells. After 2 days, lipin 3 was affinity-purified, eluted with FLAG peptide, and dialyzed (Fig. 1A). The activity and biochemistry of purified lipin 3 were investigated using 0.5 mm PA solubilized in Triton X-100 micelles (24Carman G.M. Deems R.A. Dennis E.A. Lipid signaling enzymes and surface dilution kinetics.J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 25Han 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). Lipin 3 activity was linear with time (Fig. 1B). Work by Carman et al. (24Carman G.M. Deems R.A. Dennis E.A. Lipid signaling enzymes and surface dilution kinetics.J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 25Han 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) and our laboratory (15Eaton J.M. Takkellapati S. Lawrence R.T. McQueeney K.E. Boroda S. Mullins G.R. Sherwood S.G. Finck B.N. Villén J. Harris T.E. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.J. Biol. Chem. 2014; 289: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) demonstrated that lipins 1 and 2 adhere to surface dilution kinetics. The principles of this model can be demonstrated experimentally by testing the detergent concentration-dependent changes in enzyme activity while maintaining substrate at a constant bulk concentration (24Carman G.M. Deems R.A. Dennis E.A. Lipid signaling enzymes and surface dilution kinetics.J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). We show that lipin 3 activity decreases with increasing Triton X-100 concentration, suggesting that it also adheres to surface dilution kinetics (Fig. 1C). The optimal pH for lipin 3 function was determined, with the peak activity at pH 8.0 (Fig. 1D). Maximal lipin 3 PAP activity was reached at 0.5 mm Mg2+ (Fig. 1E). The addition of 0.01 mm Mn2+, another divalent cation, increased lipin 3 activity by only 35%, relative to the maximum activity of lipin 3 at 0.5 mm Mg2+ (Fig. 1F). The PAP enzymes are characterized by their sensitivity to an alkylating agent, N-ethylmaleimide (NEM) (7Brindley D.N. Intracellular translocation of phosphatidate phosphohydrolase and its possible role in the control of glycerolipid synthesis.Prog. Lipid Res. 1984; 23: 115-133Crossref PubMed Scopus (166) Google Scholar, 26Jamal Z. Martin A. Gomez-Muñoz A. Brindley D.N. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol.J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar). The addition of 2 mm NEM resulted in a nearly complete inhibition of lipin 3 PAP activity, suggesting that it contains NEM-sensitive cysteine residues (Fig. 1G, IC50 = 0.4 mm), as is the case for lipins 1 and 2 (10Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 15Eaton J.M. Takkellapati S. Lawrence R.T. McQueeney K.E. Boroda S. Mullins G.R. Sherwood S.G. Finck B.N. Villén J. Harris T.E. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.J. Biol. Chem. 2014; 289: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 25Han 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). The addition of reducing agents stimulates the activity of lipins 1 and 2 (15Eaton J.M. Takkellapati S. Lawrence R.T. McQueeney K.E. Boroda S. Mullins G.R. Sherwood S.G. Finck B.N. Villén J. Harris T.E. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.J. Biol. Chem. 2014; 289: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 25Han 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). Indeed, 20 mm β-mercaptoethanol (β-ME) maximally stimulates lipin 3 activity (Fig. 1H). Phosphorylation has been shown to play an important role in the regulation of lipin 1 PAP activity (10Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11Huffman T.A. Mothe-Satney I. Lawrence Jr., J.C. Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1047-1052Crossref PubMed Scopus (188) Google Scholar, 14Eaton J.M. Mullins G.R. Brindley D.N. Harris T.E. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.J. Biol. Chem. 2013; 288: 9933-9945Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To investigate the phosphorylation of lipin 3, the purified protein was trypsin-digested and subjected to LC-MS/MS. Phosphorylated residues were assigned a site localization probability describing the confidence that a particular site within the sequence region is phosphorylated. We found 15 novel phosphorylation sites with localization probabilities of 0.95 or greater throughout the length of the protein (Fig. 2A and Table 1). An important phosphorylation site for lipin 1 regulation is Ser106. This site was previously identified on lipins 1 and 2 (10Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 15Eaton J.M. Takkellapati S. Lawrence R.T. McQueeney K.E. Boroda S. Mullins G.R. Sherwood S.G. Finck B.N. Villén J. Harris T.E. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.J. Biol. Chem. 2014; 289: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Although LC-MS/MS analysis did not detect well-localized phosphorylation of this site on lipin 3, a confidently identified peptide with phosphorylation on Thr105 or Ser106 was observed in lipin 3 (see Table 5 (in red) and supplemental Table S1). An important lipin protein region, enriched in phosphorylation sites, is the serine-rich domain (SRD). The SRD contains several 14-3-3 consensus-binding motifs and is important for mediating lipin 1 subcellular localization (13Péterfy M. Harris T.E. Fujita N. Reue K. Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes.J. Biol. Chem. 2010; 285: 3857-3864Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). On lipin 3, the region corresponds to amino acids Ser185–Ser230, of which Ser198, Ser218, Ser220, and Ser230 are phosphorylated with a localization probability of 0.95 or greater and Ser215 falls just short of statistically significant localization probability of 0.93 (Table 1 and supplemental Table S1). Phosphorylation on residue Ser218 is also present in lipins 1 and 2 (Ser285 in lipin 1 and Ser243 in lipin 2), and Ser287 is also phosphorylated in lipin 1 (homologous to Ser220 in lipin 3), albeit with a localization probability of 0.77 (Table 1 and supplemental Table S1) (10Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 15Eaton J.M. Takkellapati S. Lawrence R.T. McQueeney K.E. Boroda S. Mullins G.R. Sherwood S.G. Finck B.N. Villén J. Harris T.E. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.J. Biol. Chem. 2014; 289: 18055-18066Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar).Table 1Lipin 3 phosphorylation analysis by LC-MS/MSModified sequence lipin 3ResidueLocalization probabilityLGVLRS(ph)REKS601LGDSGEAFFVQELDS(ph)DEEDVPPRS941PTPES(ph)PSAQEAEEPSSQPKS1780.96PTPESPS(ph)AQEAEEPSSQPKS1800.99DIHPYS(ph)DGECTPQANLSSGDLMSPKS1981S(ph)DSELELRS2180.96SDS(ph)ELELRS2200.96LRSLEPS(ph)PLRAES2301TQNS(ph)RGAGHPPATKS3321SWS(ph)WTTPESHTPSGHPQVSRS3450.99RWS(ph)EPSNQKS4081LLES(ph)PNPEHIAECTLDSVDKS4180.99FTQHMVS(ph)YEDLTKS4600.99TEVLSSDDDVPDS(ph)PVILEVPPLPSSTPGYVPTYKKS5670.99LLFPPVVRGPSTDLAS(ph)PES8261 Open table in a new tab Table 5WT lipin 3 and lipin 3 (1PBD) phosphorylation analysis by LC-MS/MS Open table in a new tab To determine whether phosphorylation affects lipin 3 enzymatic activity, FLAG-lipin 3 was purified using affinity chromatography as described above. Before protein elution, the protein was incubated in phosphatase buffer with and without protein λ phosphatase, eluted, and dialyzed. This yielded phosphorylated (−λ) and dephosphorylated (+λ) lipin 3 (Fig. 2B). We verified that all of the phosphates were removed by overexpressing and isolating lipin 3 from 32P-radiolabeled HeLa cells expressing FLAG-lipin 3. A 30-min treatment with λ protein phosphatase in vitro removed almost all phosphates (+λ; Fig. 2C). Next, purified protein and either phosphatidylcholine (PC)/PA liposomes or Triton X-100/PA mixed micelles were used to determine whether the removal of phosphates from lipin 3 affects its basal PAP activity or affinity for substrate (Fig. 2 (D and E) and Table 2). The Kmapp values of phosphorylated (−λ) and dephosphorylated (+λ) lipin 3 for PA in liposomes were around 24.6 ± 4 and 22.5 ± 5 μm, respectively (Table 2).Table 2Steady-state kinetic data for phosphorylated (−λ) and dephosphorylated (+λ) lipin 3PEKmappKcatKcat/Kmappmol %μms−1μm−1 s−1−λ024.6 ± 40.50 ± 0.020.020−λ3037.3 ± 30.78 ± 0.120.023+λ022.5 ± 30.51 ± 0.010.023+λ3031.9 ± 90.91 ± 0.050.028 Open table in a new tab Under these conditions, the majority of PA is mono-anionic (18Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special?.Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (133) Google Scholar, 21Kooijman E.E. Tieleman D.P. Testerink C. Munnik T. Rijkers D.T. Burger K.N. de Kruijff B. An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins.J. Biol. Chem. 2007; 282: 11356-11364Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Phosphatidylethanolamine causes the pKa2 of PA to decrease to 6.9 and switches the charge of PA to −2 at physiological pH (18Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special?.Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (133) Google Scholar). The incorporation of 30 mol % PE in the PC/PA liposomes resulted in a 2-fold augmentation of phosphorylated and dephosphorylated lipin 3 PAP activity and an increase in kcat (Fig. 2D and Table 2). These data suggest that lipin 3 has greater activity toward di-anionic than mono-anionic PA independent of phosphorylation. Proximity to a hydrogen bond donor is not the only mechanism whereby PA can regulate association with to its own effector proteins. The charge of the PA headgroup can also be altered by changes in pH (14Eaton J.M. Mullins G.R. Brindley D.N. Harris T.E. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.J. Biol. Chem. 2013; 288: 9933-9945Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 21Kooijman E.E. Tieleman D.P. Testerink C. Munnik T. Rijkers D.T. Burger K.N. de Kruijff B. An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins.J. Biol. Chem. 2007; 282: 11356-11364Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). For example, lowering pH below the pKa2 of PA will result in the protonation of the headgroup due to an increase in the concentration of hydrogen ions. Conversely, an increase in pH would result in stabilization of di-anionic PA. Indeed, the activity of lipin 3 against Triton X-100/PA mixed micelles was more than 2-fold greater at pH 8.0 than at pH 7.5 (Fig. 2E). As a more direct measure of association with PA, the ability of phosphorylated and dephosphorylated Venus-tagged lipin 3 to bind PC/PA and PC/PE/PA liposomes was investigated using liposome flotation on a sucrose gradient. Venus is a yellow fluorescent protein and is easily detectable by spectrometry. It was found that in the absence of PE, the binding of lipin 3 to PC/PA liposomes was unaffected by dephosphorylation. In the presence of 30 mol % PE, the binding of lipin 3 was enhanced by 50%, regardless of its phosphorylation state (Fig. 2, F and G). These data further support phosphorylation-independent augmentation of lipin 3 PAP activity under these conditions. Lipin 1 is reported to be phosphorylated and negatively regulated upon acute insulin treatment, in a rapamycin- and Torin 1-sensitive manner (10Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr., J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11Huffman T.A. Mothe-Satney I. Lawrence Jr., J.C. Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1047-1052Crossref PubMed Scopus (188) Google Scholar, 13Péterfy M. Harris T.E. Fujita N. Reue K. Insulin-stimulated interaction with" @default.
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