title: Cholesterol 7alpha-hydroxylase is phosphorylated at multiple amino acids.
abstract: The activity of cholesterol 7alpha-hydroxylase (gpCYP7A1), the rate limiting enzyme in bile acid synthesis, has been postulated to be regulated by phosphorylation/dephosphorylation. This study has found that several kinase activators rapidly reduce the amount of bile acid produced by the human hepatoma cell line, HepG2, and that gpCYP7A1 from HepG2 cell extracts eluted in the phosphoprotein fraction of FeIII columns. After incubating the HepG2 cells with radioactive orthophosphate, the band identified as gpCYP7Al on immunoblots was strongly labeled. Recombinant gpCYP7A was expressed as 6xHIS fusion polypeptides and subjected to kinase assays. The locations of phosphorylation were mapped further by screening synthetic peptides against AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase, protein kinase A, and a panel of nine protein kinase C isoforms. AMPK, also known as 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase, phosphorylated cholesterol 7alpha-hydroxylase, suggesting a potential mechanism of coordination of cholesterol synthesis and degradation.
authors.affiliation: Department of Chemistry, Kent State University, Kent, OH 44242, USA. dstroup1@kent.edu
authors.forename: D
authors.initials: D
authors.lastname: Stroup
authors.forename: J R
authors.initials: JR
authors.lastname: Ramsaran
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D001647:Bile Acids and Salts; D011994:Recombinant Proteins; D002784:Cholesterol; D002790:Cholesterol 7-alpha-Hydroxylase