title: Regulation of FSP27 protein stability by AMPK and HSC70.
abstract: Fat-specific protein 27 (FSP27) plays a pivotal role in controlling the formation of large lipid droplet and energy metabolism. The cellular levels of FSP27 are tightly regulated through the proteasomal ubiquitin-mediated degradation. However, the upstream signals that trigger FSP27 degradation and the underlying mechanism(s) have yet to be identified. Here we show that AMP-activated protein kinase (AMPK) activation by AICAR (5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide) or phenformin induced the ubiquitination of FSP27 and promoted its degradation in 3T3-L1 adipocytes. The levels of FSP27 protein could be maintained by either knocking down AMPKα1 or blocking proteasomal pathway. Moreover, AICAR treatment induced multilocularization of LDs in 3T3-L1 adipocytes, reminiscent of the morphological changes in cells depleted of FSP27. Furthermore, mass spectrometry-based proteomic analysis identified heat shock cognate 70 (HSC70) as a novel binding protein of FSP27. The specific interaction was confirmed by co-immunoprecipitation of both ectopically expressed and endogenous proteins. Importantly, knockdown of HSC70 by small interference RNA resulted in increased half-life of FSP27 in cells treated with a protein synthesis inhibitor cycloheximide (CHX) or AICAR. However, silencing of the E3 ubiquitin ligase CHIP (COOH terminus of HSC70-interacting protein) failed to alter the stability of FSP27 protein under both conditions. Taken together, our data indicate that AMPK is a negative regulator of FSP27 stability through the proteasomal ubiquitin-dependent protein catabolic process. Promotion of FSP27 degradation may be an important factor responsible for the beneficial effect of AMPK activators on energy metabolism.
authors.affiliation: Department of Biochemistry and Molecular Biology, HEAL Program, and.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, HEAL Program, and Mayo Graduate School, Rochester, Minnesota.
authors.forename: Bradlee L
authors.initials: BL
authors.lastname: Heckmann
authors.affiliation: Department of Biochemistry and Molecular Biology, HEAL Program, and.
authors.forename: Xitao
authors.initials: X
authors.lastname: Xie
authors.affiliation: Department of Biochemistry and Molecular Biology, HEAL Program, and.
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Saarinen
authors.affiliation: Department of Biochemistry and Molecular Biology, HEAL Program, and Division of Endocrinology, Mayo Clinic in Arizona, Scottsdale, Arizona; and liu.jun@mayo.edu.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
mesh_terms: D041721:3T3-L1 Cells; D055372:AMP-Activated Protein Kinases / Q000502:physiology*; D017667:Adipocytes / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D004789:Enzyme Activation / Q000502:physiology; D055785:Gene Knockdown Techniques; D050883:HSC70 Heat-Shock Proteins / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011506:Proteins / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D050883:HSC70 Heat-Shock Proteins; C494372:Hspa8 protein, mouse; D011506:Proteins; D012265:Ribonucleotides; D025801:Ubiquitin; C525805:fat-specific protein 27, mouse; D000620:Aminoimidazole Carboxamide; D044767:Ubiquitin-Protein Ligases; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide
keywords: AMP-activated protein kinase; fat-specific protein 27; heat shock cognate 70