ser649 and ser650 are the major determinants of protein kinase a-mediated activation of human hormone-sensitive lipase against lipid substratesser649和ser650蛋白激酶的主要决定因素是一个调解的人类荷尔蒙激活脂肪酶对脂肪基质.pdfVIP

Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates 1,2 1 3 ¨ 1 2 1 Christian Krintel , Peter Osmark , Martin R. Larsen , Svante Resjo , Derek T. Logan , Cecilia Holm * 1 Division of Diabetes, Metabolism and Endocrinology, Department of Experimental Medical Science, Lund University, Lund, Sweden, 2 Department of Molecular Biophysics, Lund University, Lund, Sweden, 3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Abstract Background: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo. Methodology/Principal Findings: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type 32 enzyme. P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor ph