Tight binding inhibition of protein phosphatase-1 by phosphatidic acid. Specificity of inhibition by the phospholipid.
Phosphatidic acid (PA) has been identified as a bioactive lipid second messenger, yet despite extensive investigation, no cellular target has emerged as a mediator of its described biological effects. In this study, we identify the gamma isoform of the human protein phosphatase-1 catalytic subunit (PP1c gamma) as a high affinity in vitro target of PA. PA inhibited the enzyme dose-dependently with an IC(50) of 15 nm. Mechanistically, PA inhibited the enzyme noncompetitively with the kinetics of a tight binding inhibitor and a K(i) value of 0.97 +/- 0.24 nm. Together, these data describe one of the most potent in vitro effects of PA. To further elucidate the interaction between PA and PP1c gamma, structure/function analysis of the lipid was carried out using commercially available and synthetically generated analogs of PA. These studies disclosed that the lipid-protein interaction is dependent on the presence of the lipid phosphate as well as the presence of the fatty acid side chains, because lipids lacking either of these substituents resulted in complete loss of inhibition. However, the specific composition of the fatty acid side chains was not important for inhibition. Using 1-O-hexadecyl,2-oleoyl-PA, it was also shown that the carbonyl group of the sn-1 acyl linkage is not required for the lipid-protein interaction. Finally, using a lipid-protein overlay assay, it was demonstrated that PP1c gamma specifically and directly interacts with phosphatidic acid while not significantly binding other phospholipids. These results identify PA as a tight binding and specific inhibitor of PP1, and they raise the hypothesis that PP1c gamma may function as a mediator of PA action in cells. They also argue for the existence of a specific high affinity PA-binding domain on the enzyme.
Jones
Jeffrey A
JA
Molecular and Cellular Biology & Pathobiology Program and the Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Hannun
Yusuf A
YA
D004926:Escherichia coli; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology; D004676:Myelin Basic Protein / Q000378:metabolism; D010712:Phosphatidic Acids / Q000494:pharmacology*; D051966:Phospholipase C gamma; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010762:Phosphorylase a / Q000378:metabolism; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D007527:Isoenzymes; D008246:Lysophospholipids; D004676:Myelin Basic Protein; D010712:Phosphatidic Acids; D011994:Recombinant Proteins; D010762:Phosphorylase a; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D010738:Type C Phospholipases; D051966:Phospholipase C gamma