Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis.

Abstract

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser433, Ser492, and Ser517). Peri 301-517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.