Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro.
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.
Anthonsen
M W
MW
Department of Cell and Molecular Biology, Section for Molecular Signaling, Lund University, S-221 00 Lund, Sweden.
Rönnstrand
L
L
Wernstedt
C
C
Degerman
E
E
Holm
C
C
D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D007545:Isoproterenol / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D002787:Sterol Esterase / Q000235:genetics / Q000378:metabolism*
D002787:Sterol Esterase; D007545:Isoproterenol