The phosphorylation of serine 492 of perilipin A facilitates ATGL-mediated lipolysis by CGI-58 independent mechanisms

Perilipin A is a lipid droplet-associated protein that controls triacylglycerol storage and hydrolysis in adipocytes. Lipolysis of triacylglycerols by adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) is enabled by the phosphorylation of perilipin A by protein kinase A (PKA) on six serine residues. The main goal of this study was to understand how phosphorylation of serine 492 of perilipin A promotes lipolysis by ATGL, with or without the co-activator CGI-58. We used NIH 3T3 CAR? fibroblasts ectopically expressing mutated variants of perilipin and ATGL with or without CGI-58. The perilipin variants included wild-type perilipin A, perilipin with serine 492 mutated to alanine (perilipin S492A), perilipin in which only serine 492 can be phosphorylated (perilipin Not5), and perilipin in which none of the serine residues within PKA sites can be phosphorylated (perilipin All6). Cells expressing these perilipins and ATGL, with or without CGI-58, were lipid loaded with radioactive oleate to label triacylglycerol; lipolysis was assessed by measuring the appearance of radiolabeled oleate in the culture medium under basal conditions and when lipolysis was stimulated by the addition of forskolin and isobutylmethylxanthine.;Rates of basal lipolysis were similar in cells expressing all perilipin variants and significantly lower than rates of stimulated lipolysis. Stimulated ATGL-mediated lipolysis was comparable in cells expressing perilipin Not5 and cells expressing wild-type perilipin A, whereas cells expressing perilipin S492A showed decreased rates of lipolysis. Comparable maximal rates of stimulated lipolysis were observed in cells expressing either perilipin Not5 or wild-type perilipin A when both ATGL and CGI-58 were expressed, whereas cells expressing perilipin S492A showed a trend towards reduced lipolysis. We conclude that the phosphorylation of serine 492 of perilipin A is required for maximal lipolysis catalyzed by ATGL. In the absence of serine 492, the phosphorylation of serine residues within PKA sites 1-4 and 6 can also facilitate ATGL-mediated lipolysis, although not to maximal levels. Since similar results were obtained with and without CGI-58, we propose that phosphorylation of serine 492 facilitates lipolysis by a general mechanism that is not dependent on the availability of CGI-58 to activate ATGL.