Abstract
Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70-100 and 80-90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130-140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D(3) enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-(33)P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55-58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.
Highlights
UDP-glucuronosyltransferase 2B15 (UGT2B15) is the only 5␣-dihydrotestosterone (DHT)-metabolizing enzyme in prostate luminal cells
It was of interest to extend the curcumin studies by focusing on the predicted phosphorylation sites in luminal cell-distributed 2B15, which is critical for DHT homeostasis in this key cell, which carries out DHT-occupied androgen receptor-dependent prostate-specific functions
Because down-regulation and recovery following exposure to 10 M curcumin did not differ in the presence and absence of cycloheximide (Fig. 2), the results indicate that the effect was independent of protein synthesis and was likely dependent on disrupted phosphate signaling (25)
Summary
UDP-glucuronosyltransferase 2B15 (UGT2B15) is the only 5␣-dihydrotestosterone (DHT)-metabolizing enzyme in prostate luminal cells. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5␣-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions. Electron micrographs that show a one-to-one stratified arrangement of basal/luminal cells attached to basement membrane in human prostate (Fig. 1) (6) support a model of exclusive movement of small molecules from blood to basal cells and, in turn, to luminal cells These considerations indicate that prostate DHT regulation is necessarily complex and is interdependent upon the two epithelial cell types. Questions arise as to whether phosphorylation of 2B15 likely impacts DHT clearance
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