Phosphorylation of a UDP-glucuronosyltransferase regulates substrate specificity.
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-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 after exposure to curcumin or calphostin-C indicated that the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKCepsilon-specific inhibitor peptide supported PKC involvement. Co-immunoprecipitation, colocalization by means of immunofluorescence, and cross-linking studies of PKCepsilon and UGT1A7His revealed that the proteins reside within 11.4 angstroms of each other. Moreover, mutation of three PKC sites in each UGT isozyme demonstrated that 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 17beta-estradiol. S432G-UGT1A10 exhibited a minor pH shift without substrate alterations. PKCepsilon involvement was confirmed by the demonstration that PKCepsilon overexpression enhanced activity of UGT1A7 but not of its S432 mutant and the conversion of 17beta-[14C]estradiol by S432G-UGT1A7 but not by UGT1A7. Consistent with these observations, treatment of UGT1A7-transfected cells with PKCepsilon-specific inhibitor peptide or general PKC inhibitors increased 17beta-estradiol catalysis between 5- and 11-fold, with parallel decreases in phosphoserine-432. Here, we report a mechanism involving PKC-mediated phosphorylation of UGT such that phosphoserine/threonine regulates substrate specificity in response to chemical exposures, which possibly confers survival benefit.
Basu
Nikhil K
NK
Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 9S-241, Bethesda, MD 20892-1830, USA.
Kovarova
Martina
M
Garza
Amanda
A
Kubota
Shigeki
S
Saha
Tapas
T
Mitra
Partha S
PS
Banerjee
Rajat
R
Rivera
Juan
J
Owens
Ida S
IS
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002250:Carbon Radioisotopes; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003474:Curcumin / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D007527:Isoenzymes; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D009281:Naphthalenes / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism; D014162:Transfection
D002250:Carbon Radioisotopes; D007527:Isoenzymes; D009281:Naphthalenes; D010768:Phosphoserine; D013912:Threonine; D004958:Estradiol; D014453:Glucuronosyltransferase; C107611:UDP-glucuronosyltransferase, UGT1A7; D011493:Protein Kinase C; C058819:calphostin C; D003474:Curcumin