Abstract
BackgroundHormone-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 FindingsIn 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. 32P-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 S0.5 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 S0,5 using the TO substrate.ConclusionsOur 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.
Highlights
Fatty acids mobilized from stored triacylglycerols are a major energy source in humans
Our results demonstrate that protein kinase A (PKA) activates human Hormone-sensitive lipase (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
Summary
Fatty acids mobilized from stored triacylglycerols are a major energy source in humans. Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Effects of point mutations on the activity of HSL Mutating Ser554 to alanine (the SVA-SS variant) resulted in a slight decrease in lipolytic activity. Mimicking the charge effect of an anti-lipolytic phosphorylation of the basal site by mutating Ser554 to glutamic acid (the SVE-SS variant) resulted in similar to wild type activity. Simultaneous mutation of both Ser552 and Ser554 to alanine (the AVA-SS variant) had no effect on the activity. Mutating all four serines, including Ser649 and Ser650 (the AVA-AA variant) decreased the activity of human HSL against TO, MOME and CO substrates by 67%, 63% and 60%, respectively. The activity against pNPB decreased, but only by 30% compared to wild type HSL (Figure 4)
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