Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro

J Biol Chem. 1998 Jan 2;273(1):215-21. doi: 10.1074/jbc.273.1.215.


Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of 32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser --> Ala mutations in residues 563 and 565 (S563A, S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675 phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipocytes / drug effects
  • Adipocytes / enzymology
  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cells, Cultured
  • Electrophoresis, Gel, Two-Dimensional
  • Enzyme Activation
  • Isoproterenol / pharmacology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Mapping
  • Phosphorylation
  • Rats
  • Rats, Sprague-Dawley
  • Sterol Esterase / genetics
  • Sterol Esterase / metabolism*


  • Sterol Esterase
  • Isoproterenol