title: Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates.
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.
  
  
  RESULTS
  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 enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate.
  
  
  CONCLUSIONS
  Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region.
authors.affiliation: Division of Diabetes, Metabolism and Endocrinology, Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Christian
authors.initials: C
authors.lastname: Krintel
authors.forename: Peter
authors.initials: P
authors.lastname: Osmark
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Svante
authors.initials: S
authors.lastname: Resjö
authors.forename: Derek T
authors.initials: DT
authors.lastname: Logan
authors.forename: Cecilia
authors.initials: C
authors.lastname: Holm
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005227:Fatty Acids / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D008055:Lipids / Q000737:chemistry*; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D002787:Sterol Esterase / Q000737:chemistry / Q000378:metabolism*; D014280:Triglycerides / Q000737:chemistry
chemical_list: D005227:Fatty Acids; D008055:Lipids; D014280:Triglycerides; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D002787:Sterol Esterase