title: Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.
abstract: Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver.
authors.affiliation: Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A. Department of Pharmacology, College of Medicine, the University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, U.S.A.
authors.forename: Qingming
authors.initials: Q
authors.lastname: Dong
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, the University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163, U.S.A.
authors.forename: Francesco
authors.initials: F
authors.lastname: Giorgianni
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, the University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163, U.S.A.
authors.forename: Sarka
authors.initials: S
authors.lastname: Beranova-Giorgianni
authors.affiliation: Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A. Department of Pharmacology, College of Medicine, the University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, U.S.A.
authors.forename: Xiong
authors.initials: X
authors.lastname: Deng
authors.affiliation: Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
authors.forename: Robert N
authors.initials: RN
authors.lastname: O'Meally
authors.affiliation: Department of Physiology, College of Medicine, the University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163, U.S.A. Children's Foundation Research Institute, Le Bonheur Children's Hospital, Department of Pediatrics, the University of Tennessee Health Science Center, 50 North Dunlap, Memphis, TN 38103, U.S.A.
authors.forename: Dave
authors.initials: D
authors.lastname: Bridges
authors.affiliation: Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A. Department of Pharmacology, College of Medicine, the University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, U.S.A.
authors.forename: Edwards A
authors.initials: EA
authors.lastname: Park
authors.affiliation: Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Cole
authors.affiliation: Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A. Department of Pharmacology, College of Medicine, the University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, U.S.A.
authors.forename: Marshall B
authors.initials: MB
authors.lastname: Elam
authors.affiliation: Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A. Department of Pharmacology, College of Medicine, the University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, U.S.A. rraghow@uthsc.edu.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Raghow
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008055:Lipids / Q000096:biosynthesis* / Q000235:genetics; D008099:Liver / Q000378:metabolism*; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D059748:Proteolysis; D051381:Rats; D051780:Sterol Regulatory Element Binding Protein 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D008055:Lipids; C497387:SREBF1 protein, human; C497389:Srebf1 protein, rat; D051780:Sterol Regulatory Element Binding Protein 1; D038362:Glycogen Synthase Kinase 3; D046988:Proteasome Endopeptidase Complex
keywords: GSK-3; cdc4 phosphodegron (CPD); mass spectrometry (MS); phosphorylation; sterol regulatory element binding protein (SREBP); ubiquitination