Identification of Novel Phosphorylation Sites in Hormone-sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro

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 32 P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser 3 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 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-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.Free fatty acids stored as triacylglycerols in the adipocytes comprise the quantitatively most important energy substrate in mammals. Hormone-sensitive lipase (HSL) 1 (EC 3.1.1.3) catalyzes the first and rate-limiting step in the hydrolysis of stored triacylglycerols and is thereby a key enzyme in the mobilization of free fatty acids from adipose tissue (1). The hormonal and neural control of lipolysis is exerted by regulation of HSL activity, mediated by reversible phosphorylation (2, 3). In response to catecholamines and other fast-acting lipolytic hormones, HSL is activated through the phosphorylation by protein kinase A (PKA). The major antilipolytic hormone insulin prevents cAMP-mediated phosphorylation and activation of HSL (3). The antilipolytic effect of insulin is brought about mainly by activation of phosphodiesterase 3B (4).HSL has in intact rat adipocytes been reported to be phosphorylated at two sites (3). These sites were later identified as Ser-563 (5) and Ser-565 (6). In hormonally quiescent cells, only Ser-565 is phosphorylated (3). Stimulation with noradrenaline increases the phosphorylation extent of Ser-563 to that of Ser-565 (3). Ser-563 is also phosphorylated in vitro by PKA (5). Ser-565 has been shown to be phosphorylated in vitro by the 5Ј-AMP-activated kinase (6). This kinase, which phosphorylates and regulates the activity of other key enzymes in lipid metabolism (7), acetyl-CoA carboxylase (fatty acid synthesis), and 3-hydroxy-3-methylglutaryl-CoA (cholesterol synthesis), has also been suggested ...

Triacylglycerol lipases and metabolic control: implications for health and disease

“…1). Although it was known for several years that PKA increased HSL activity through a phosphorylation-dependent mechanism, mutagenesis experiments demonstrated dependence for this effect on phosphorylation at Ser 563 , Ser 659 , and Ser 660 (2,67). However, it should be noted that there is some doubt as to the importance of Ser 563 (2).…”

“…Although it was known for several years that PKA increased HSL activity through a phosphorylation-dependent mechanism, mutagenesis experiments demonstrated dependence for this effect on phosphorylation at Ser 563 , Ser 659 , and Ser 660 (2,67). However, it should be noted that there is some doubt as to the importance of Ser 563 (2). The PKA phosphorylation of HSL results in modest activation of the enzyme (2-to 3-fold) and promotes the translocation of HSL to the LD (21), an effect that is critical for enhancing lipolytic capacity as a result of ␤-adrenergic stimulation.…”

Insulin and hyperandrogenism in women with polycystic ovary syndrome

“…These modulations require an increase in the transcription factor steroid regulatory element-binding protein (SREBP)-1c [55]. In adipocytes, insulin also inhibits lipolysis through inhibition of the enzyme hormone-sensitive lipase (HSL) [56]. Recent data showed that protein kinase B (PKB) is required for the regulation of lipogenesis and for the antilipolysis effect of insulin [57].…”

Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes

“…However, studies performed on a purified preparation of the catalytic subunit of PKA revealed dose-dependent inhibition by vanadium [36]. The decrease of isoprenaline-induced PKA activation observed in response to vanadate and a low concentration of pV would presumably lead to a lowering of PKA-mediated phosphorylation and activation of hormone-sensitive lipase [37] that could explain the anti-lipolytic effect of these agents. However, a direct effect of these agents on hormone-sensitive lipase also cannot be excluded.…”