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.

Mutations in the gene encoding comparative gene identifi cation-58 [CGI-58; also ␣ / ␤ hydrolase domain 5 (ABHD5)] cause Chanarin-Dorfman syndrome (CDS) ( 1 ), a neutral lipid storage disorder characterized by excessive accumulation of triacylglycerols (TAGs) in cells and tissues, including liver, skeletal muscle, intestinal epithelia, leukocytes, keratinocytes, and skin fi broblasts, leading to hepatomegaly, ichthyosis, and mild muscle weakness ( 2-7 ). These early observations suggested that CGI-58 plays an important role in TAG homeostasis in multiple tissues. Although CGI-58 is a member of the lipase subfamily of ␣ / ␤ hydrolase domain proteins, it lacks a serine residue in a conserved sequence (GXSXG) that normally harbors the nucleophilic component of the catalytic triad ( 1 ). Identifi cation of CGI-58 as a coactivator of the widely expressed adipose TG lipase (ATGL) ( 8 ) provided a mechanistic explanation for the phenotype of CDS patients; loss of functional CGI-58 reduces ATGL-mediated hydrolysis of TAGs, in turn increasing TAG storage in tissues. Individuals with mutations in ATGL show similarities to CDS patients with TAG accumulation in various tissues including liver and skeletal muscle ( 9 ); however, these individuals lack ichthyosis and have more severe skeletal muscle myopathy than individuals with CDS, as well as cardiomyopathy. The phenotypic differences between individuals with mutations in CGI-58 and those with mutations in ATGL suggest that CGI-58 serves one or more functions distinct from coactivation of ATGL.Abstract Mutations in the gene encoding comparative gene identifi cation 58 (CGI-58)/ ␣ / ␤ hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identifi ed as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affi nity purifi cation procedures, plsC, the sole LPAAT in Escherichia coli . Purifi cation protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S -transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI (3)

Comparative gene identification‐58 (CGI‐58) interacts with and co‐activates adipose triglyceride lipase (ATGL). The H82R mutation in human CGI‐58 causes a neutral lipid storage disorder (NLSD) characterized by ichthyosis and excessive triglyceride storage in many types of cells. We studied the comparable H84R mutation in mouse CGI‐58, and H84A mutated CGI‐58, to ask how CGI‐58 function is impaired. The ectopic expression of wild‐type (WT) CGI‐58 in human NLSD fibroblasts reduced excessive triglyceride storage to normal levels, whereas H84R CGI‐58 was significantly less effective. Additionally, neither H84R nor H84A CGI‐58 co‐activated ATGL in an in vitro triglyceride hydrolase assay. A co‐immunoprecipitation assay demonstrated that H84R and H84A CGI‐58 bound ATGL as well as WT CGI‐58. Immunofluorescence microscopy revealed that H84R and H84A CGI‐58 localized to ectopic perilipin 1A on lipid droplets of cultured NIH3T3CARΔ fibroblasts as well as WT CGI‐58. Moreover, the addition of forskolin and isobutylmethylxanthine to the cells triggered the dispersion of all three variants of CGI‐58 from the perilipin scaffold into the cytoplasm. Thus, H84R CGI‐58 shows appropriate subcellular localization in cells expressing perilipin 1A and binds to ATGL, yet does not co‐activate hydrolase activity. Future experiments will explore additional mechanisms for deficient CGI‐58 function. Grant Funding Source: Supported by NIH R01 DK054797.