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- Anthonsen, M W, et al.
(författare)
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Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro
- 1998
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 273:1, s. 215-221
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Tidskriftsartikel (refereegranskat)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.
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- Rahn, Tova, et al.
(författare)
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Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol
- 1996
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 271:19, s. 11575-11580
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Tidskriftsartikel (refereegranskat)abstract
- Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).
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