Control of Adipose Triglyceride Lipase Action by Serine 517 of Perilipin A Globally Regulates Protein Kinase A-stimulated Lipolysis in Adipocytes

• DOI: 10.1074/jbc.m605770200
• Publication Date: 2006-11-19T01:12:33Z
• Authors: Hideaki Miyoshi; James W. Perfield; Sandra C. Souza; Wen-Jun Shen; Hui-Hong Zhang; Zlatina S. Stancheva; Fredric B. Kraemer; Martin S. Obin; Andrew S. Greenberg
• Keywords: 0301 basic medicine; Perilipin-1; 493.12; Lipolysis; Lipase; Fibroblasts; Phosphoproteins; Cyclic AMP-Dependent Protein Kinases; Mice, Mutant Strains; Adenoviridae; Cell Line; Protein Structure, Tertiary; Mice; 03 medical and health sciences; Adipocytes; Mutagenesis, Site-Directed; Serine; Animals; Phosphorylation; Carrier Proteins; Carboxylic Ester Hydrolases

Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the "downstream" lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a "phosphomimetic" (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel "master regulator" of PKA-stimulated adipocyte lipolysis.