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 ...

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Double-stranded RNA (dsRNA) is produced during the replication cycle of most viruses and triggers antiviral immune responses through Toll-like receptor 3 (TLR3). However, the molecular mechanisms and subcellular compartments associated with dsRNA-TLR3-mediated signaling are largely unknown. Here we show that c-Src tyrosine kinase is activated by dsRNA in human monocyte-derived dendritic cells, and is recruited to TLR3 in a dsRNAdependent manner. DsRNA-induced activation of interferon-regulatory factor 3 and signal transducer and activator of transcription 1 was abolished in Src kinase-deficient cells, and restored by adding back c-Src, suggesting a central role of c-Src in antiviral immunity. We also provide evidence that TLR3 is localized in the endoplasmic reticulum of unstimulated cells, moves to dsRNA-containing endosomes in response to dsRNA, and colocalizes with c-Src on endosomes containing dsRNA in the lumen. These results provide novel insight into the molecular mechanisms of TLR3-mediated signaling, which may contribute to the understanding of innate immune responses during viral infections.