title: Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins.
abstract: Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.
authors.affiliation: Geriatric Research, Education, and Clinical Center, Baltimore Veterans Affairs Health Care Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Liping
authors.initials: L
authors.lastname: Hu
authors.forename: Knut
authors.initials: K
authors.lastname: Dalen
authors.forename: Heidi
authors.initials: H
authors.lastname: Dorward
authors.forename: Amy
authors.initials: A
authors.lastname: Marcinkiewicz
authors.forename: Deanna
authors.initials: D
authors.lastname: Russell
authors.forename: Dawei
authors.initials: D
authors.lastname: Gong
authors.forename: Constantine
authors.initials: C
authors.lastname: Londos
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Cecilia
authors.initials: C
authors.lastname: Holm
authors.forename: Megan A
authors.initials: MA
authors.lastname: Rizzo
authors.forename: Dawn
authors.initials: D
authors.lastname: Brasaemle
authors.forename: Carole
authors.initials: C
authors.lastname: Sztalryd
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins; D006224:Cricetinae; D003412:Cricetulus; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D008066:Lipolysis; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000070778:Perilipin-1; D000070780:Perilipin-2; D000070797:Perilipin-5; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D002787:Sterol Esterase / Q000378:metabolism*; D014280:Triglycerides / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C000602982:PLIN2 protein, human; D000070778:Perilipin-1; D000070780:Perilipin-2; D000070797:Perilipin-5; D010750:Phosphoproteins; C000602983:Plin2 protein, mouse; C511805:Plin5 protein, mouse; D011506:Proteins; D014280:Triglycerides; D002787:Sterol Esterase