UDP-glucuronosyltransferase (UGT) isozymes catalyze detoxification of numerous chemical toxins present in our daily diet and environment by conjugation to glucuronic acid. The special properties and enzymatic mechanism(s) that enable endoplasmic reticulum (ER)-bound UGT isozymes to convert innumerable structurally diverse lipophiles to excretable glucuronides are unknown. Inhibition of cellular UGT1A7 and UGT1A10 activities and of [33P]orthophosphate incorporation into immunoprecipitable proteins following exposure to curcumin or calphostin-C indicate the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKCepsilon-specific inhibitor peptide support PKC involvement. Computer analysis revealed each UGT isozyme has from 4 to 6 PKC sites. Co-immunoprecipitation, co-localization via immunofluorescence and cross-linking studies of PKCepsilon and UGT1A7His revealed the proteins reside within 11.4 Angstrom of each other. Mutation of three PKC sites in each UGT isozyme demonstrated T73A/G and T202A/G caused null activity, whereas S432G-UGT1A7 caused a major shift of its pH-8.5 optimum to 6.4 with new substrate selections, including 17b-estradiol. PKCepsilon involvement was confirmed by (a) the demonstration that PKCepsilon overexpression enhanced activity of UGT1A7 but not that of its S432 mutant and (b) the conversion of 17Beta-[14C]estradiol by S432G-1A7 but not by 1A7. Consistent with these observations, treatment of 1A7-transfected cells with PKCepsilon-specific inhibitor peptide or general PKC inhibitors dramatically increased 17b-estradiol catalysis with parallel decreases in phospho-serine-432. Thus, protein kinase C-mediated phosphorylation of serine/threonine in UGT regulates substrate specificity, which possibly confers survival benefit.
