IDENTIFICATION OF NOVEL PHOSPHORYLATION SITES IN HORMONE-SENSITIVE LIPASE THAT ARE PHOSPHORYLATED IN RESPONSE TO ISOPROTERENOL AND GOVERN ACTIVATION PROPERTIES IN-VITRO
Mw. Anthonsen et al., IDENTIFICATION OF NOVEL PHOSPHORYLATION SITES IN HORMONE-SENSITIVE LIPASE THAT ARE PHOSPHORYLATED IN RESPONSE TO ISOPROTERENOL AND GOVERN ACTIVATION PROPERTIES IN-VITRO, The Journal of biological chemistry, 273(1), 1998, pp. 215-221
Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysi
s, Stimulation of rat adipocytes with isoproterenol results in phospho
rylation of HSL and a 50-fold increase in the rate of lipolysis, In th
is study, we used site-directed mutagenesis and two-dimensional phosph
opeptide mapping to show that phosphorylation sites other than the pre
viously identified Ser-563 are phosphorylated in HSL in response to is
oproterenol stimulation of P-32-labeled rat adipocytes, Phosphorylatio
n of HSL in adipocytes in response to isoproterenol and in vitro phosp
horylation of HSL containing Ser --> Ala mutations in residues 563 and
565 (S563A,S565A) with protein kinase A (PKA), followed by tryptic ph
osphopeptide mapping resulted in two tryptic phosphopeptides, These tr
yptic phosphopeptides co-migrated with the phosphopeptides released by
the same treatment of F(654)HPRRSSQGVLHMPLYSSPIVK(675) phosphorylated
with PKA, Analysis of the phosphorylation site mutants, S659A, S660A,
and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-66
0 was necessary to abolish the activation of HSL toward a triolein sub
strate 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 previousl
y identified Ser-563, two other PKA phosphorylation sites, Ser-659 and
Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-66
0 are the major activity controlling sites in vitro.