| Title : Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro - Anthonsen_1998_J.Biol.Chem_273_215 |
| Author(s) : Anthonsen MW , Ronnstrand L , Wernstedt C , Degerman E , Holm C |
| Ref : Journal of Biological Chemistry , 273 :215 , 1998 |
| Abstract : 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. |
| ESTHER : Anthonsen_1998_J.Biol.Chem_273_215 |
| PubMedSearch : Anthonsen_1998_J.Biol.Chem_273_215 |
| PubMedID: 9417067 |
Anthonsen MW, Ronnstrand L, Wernstedt C, Degerman E, Holm C (1998)
Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro
Journal of Biological Chemistry
273 :215
Anthonsen MW, Ronnstrand L, Wernstedt C, Degerman E, Holm C (1998)
Journal of Biological Chemistry
273 :215