Abstract
CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS239S240, we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immunoblotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.
Highlights
IntroductionUpon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation
To test the hypothesis that CGI-58 is a substrate for protein kinase A (PKA), we studied the phosphorylation of recombinant mouse CGI-58 that was purified from E. coli lysates [19]
Recombinant CGI-58 was incubated with mammalian PKA and [␥-32P]ATP in vitro; the incorporation of radioactive phosphate into CGI-58 was monitored by phosphorimaging analysis of blots and revealed that recombinant CGI-58 is a substrate for PKA (Fig. 1B)
Summary
Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58 Both CGI-58 and S239A/S240Amutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.—Sahu-Osen, A., G. Hormone binding triggers a G protein-mediated cascade that activates adenylyl cyclase, increasing levels of cAMP, in Abbreviations: ATGL, adipose triglyceride lipase; IBMX, isobutylmethylxanthine; NLSD, neutral lipid storage disorder; PKA, protein kinase A or cAMP-dependent protein kinase; PVDF, polyvinylidene difluoride; TCEP, Tris(2-carboxyethyl)phosphine; TEV, tobacco etch virus
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