Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity.
Our discovery of rapid down-regulation of human bilirubin UDP-glucuronosyltransferase (UGT) in colon cell lines that was transient and irreversible following curcumin- and calphostin-C-treatment, respectively, suggested phosphorylation event(s) were involved in activity. Likewise, bilirubin-UGT1A1 expressed in COS-1 cells was inhibited by curcumin and calphostin-C. Because calphostin-C is a highly specific protein kinase C (PKC) inhibitor, we examined and found 4 to 5 predicted PKC phosphorylation sites in 11 UGTs examined. UGT1A1 incorporated [33P]orthophosphate, which was inhibited by calphostin-C. Also triple mutant, T75A/T112A/S435G-UGT1A1, at predicted PKC sites failed to incorporate [33P]orthophosphate. Individual or double mutants exhibited dominant-negative, additive, or no effect, while the triple mutant retained 10-15% activity towards bilirubin and two xenobiotics. Compared to wild-type, S435G and T112A/S435G shifted pH-optimum for eugenol, but not for bilirubin or anthraflavic acid, toward alkaline and acid conditions, respectively. This represents the first evidence that a UGT isozyme requires phosphorylation for activity.
Basu
Nikhil K
NK
Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1830, USA.
Kole
Labanyamoy
L
Owens
Ida S
IS
D000595:Amino Acid Sequence; D000818:Animals; D001663:Bilirubin / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D003474:Curcumin / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005966:Glucuronidase / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014407:Tumor Cells, Cultured; D000099148:UGT1A1 Enzyme
D001663:Bilirubin; D003474:Curcumin; D004791:Enzyme Inhibitors; D005966:Glucuronidase; D014453:Glucuronosyltransferase; D009281:Naphthalenes; D011493:Protein Kinase C; D011994:Recombinant Proteins; D000099148:UGT1A1 Enzyme; C058819:calphostin C