Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro
Författare
Summary, in English
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.
Avdelning/ar
Publiceringsår
1998
Språk
Engelska
Sidor
215-221
Publikation/Tidskrift/Serie
Journal of Biological Chemistry
Volym
273
Issue
1
Dokumenttyp
Artikel i tidskrift
Förlag
American Society for Biochemistry and Molecular Biology
Ämne
- Endocrinology and Diabetes
Status
Published
Forskningsgrupp
- Insulin Signal Transduction
- Molecular Endocrinology
ISBN/ISSN/Övrigt
- ISSN: 1083-351X