Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity

Biochem Biophys Res Commun. 2003 Mar 28;303(1):98-104. doi: 10.1016/s0006-291x(03)00241-9.


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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bilirubin / metabolism
  • Binding Sites
  • Blotting, Western
  • COS Cells
  • Catalysis
  • Curcumin / metabolism
  • Curcumin / pharmacology
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Glucuronidase / metabolism
  • Glucuronosyltransferase / chemistry*
  • Glucuronosyltransferase / metabolism*
  • Humans
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Naphthalenes / metabolism
  • Phosphorylation
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Time Factors
  • Tumor Cells, Cultured


  • Enzyme Inhibitors
  • Naphthalenes
  • Recombinant Proteins
  • UGT1A1 enzyme
  • Glucuronosyltransferase
  • Protein Kinase C
  • Glucuronidase
  • calphostin C
  • Curcumin
  • Bilirubin