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.[1]References
- Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity. Basu, N.K., Kole, L., Owens, I.S. Biochem. Biophys. Res. Commun. (2003) [Pubmed]
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