Regulated phosphorylation of a major UDP-glucuronosyltransferase isozyme by tyrosine kinases dictates endogenous substrate selection for detoxification

Abstract

Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE1) with barely detectable 17β-estradiol (E2) conversion following expression in COS-1 cells. Consistent with the UDP-glucuronosyltransferase requirement for regulated phosphorylation, we discovered that 2B7 requires Src-dependent tyrosine phosphorylation. Y236F-2B7 and Y438F-2B7 mutants were null and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [ 33P]orthophosphate and that 2B7His, previously expressed in SYF-(Src,Yes,Fyn)-/- cells, was Src-supported or phosphorylated under in vitro conditions. Unexpectedly, 2B7 expressed in SYF-/- and SYF+/- cells metabolized 4-OHE1 at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E2 metabolism was 16- and 9-fold higher than in COS-1. Because anti-Tyr(P)-438-2B7 detected Tyr(P)-438-2B7 in each cell line, results indicated that unidentified tyrosine kinase(s) (TKs) phosphorylated 2B7 in SYF -/-. 2B7-transfected COS-1 treated with increasing concentrations of the Src-specific inhibitor PP2 down-regulated 4-OHE1 glucuronidation reaching 60% maximum while simultaneously increasing E2 metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E2 metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF-/- is more competent at metabolizing E2 in cellulo than 2B7 expressed in COS-1. To confirm Src-controlled 2B7 prevents toxicity, we showed that 2B7-transfected COS-1 efficiently protected against 4-OH-E1-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE1, but not E2, in COS-1, whereas non-Src-phosphorylated 2B7 metabolizes both chemicals. Importantly, we determined that 2B7 substrate selection is not fixed but varies depending upon the TK(s) that carry out its required phosphorylation.