title: Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.
abstract: The lipin gene family encodes a class of Mg(2+)-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.
authors: Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908.
authors: James M
authors: JM
authors: Eaton
authors: Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908.
authors: Garrett R
authors: GR
authors: Mullins
authors: Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton T6G 2S2, Canada.
authors: David N
authors: DN
authors: Brindley
authors: Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908. Electronic address: teh3c@virginia.edu.
authors: Thurl E
authors: TE
authors: Harris
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008823:Micelles; D017830:Octoxynol / Q000494:pharmacology; D010711:Phosphatidate Phosphatase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D010712:Phosphatidic Acids / Q000737:chemistry*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D055672:Static Electricity; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D003902:Detergents; D008081:Liposomes; D008823:Micelles; D010712:Phosphatidic Acids; D011994:Recombinant Proteins; D017830:Octoxynol; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; C485999:LPIN1 protein, human; D010711:Phosphatidate Phosphatase; D008274:Magnesium