Whereas COS-1 cell-expressed human UDP-glucuronosyltransferase (UGT)-2B17 metabolizes primarily dihydrotestosterone (DHT) and its androstane-diol (ADT-DIOL) metabolite, we have evidence the prostate-distributed isozyme requires regulated phosphorylation by its reversible and irreversible inhibition following treatment with curcumin or general PKC inhibitors, respectively. The fact that treatment of COS 1-expressed 2B17 with PKCalpha-siRNA downregulates activity or treatment with PKCepsilon-siRNA, Src-siRNA or Src kinase-specific PP2 inhibitor upregulates its activity indicates PKCalpha activates 2B17 and that PKCepsilon and/or Src downregulate activity. PKC-site mutants--S172A-2B17 and S422A-2B17-- are null and unchanged, respectively; tyrosine kinase-site mutants --Y99F-2B17 and Y237F-2B17-- show preferential inhibition of DHT over ADT-diol glucuronidation at the 70/30 and 90/60 percent levels, respectively-- a phenomenon that converts to essentially 50 to 60/70 percent following expression in Src-free cells. Solubilized 2B17-His-transfected Src-free fibroblasts subjected to in-vitro 33PATP-dependent phosphorylation by PKCepsilon and/or Src followed by affinity purification, and SDS-gel analyzed revealed higher radiolabeling of 55- to 58-kDa 2B17-His compared to alpha and/or Src. Activity for all combined kinases reactions were additive. Collectively, evidence indicates 2B17 requires regulated phosphorylation by PKCalpha, PKCepsilon and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Evidence indicates that the 95% identical prostate basal cell-distributed UGT2B17 and luminal cell-distributed 2B15 are overall inversely programmed by phosphorylation. Related manuscript: Combined positive PKCalpha and negative PKCepsilon and Src effects on regulated phosphorylation of human dihydrotestosterone-metabolizing UGT-2B17 depress its constitutive activity-- N.K. Basu, M. Basu, J. Park, R. Banerjee and I. S. Owens, J. Biol. Chem. 2B17 Under Revision- Human prostate basal cell distributed UDP-glucuronosyltransferase-2B17 (UGT-2B17) and its 97%-identical UGT-2B15 homolog metabolize both dihydrotestosterone (DHT) and its androstane- diol (Andro) metabolite. Mass spectrometry has confirmed UGT-2B17 has 4 of 5 predicted phosphorylation sites in luminal-cell-distributed UGT-2B15; dissimilarly, 2B17 contains the rare tri-phosphorylated-TYS sequence at position 98-100. Both isozymes undergo regulated phosphorylation for two different functions. Like UGT-2B15, anti-PKCalpha-immunocomplexed UGT-2B17-S172 confirmed phosphate-signaling enables a non-fixed active-site that catalyzes an unspecified number of substrates based on analysis of 6/19 human UGTs. Contrariwise, anti-PKCepsilon immnocomplexes of 2B17 generated dense smearing patterns, except for its Y99F mutant or following expression of constructs in Src-free cells. Hence, previously identified robust and critical signaling by the Src/PKCepsilon-partnership phosphorylation site, TYS, located at position 98-100 in 2B17 suggests its critical role. Following 2B17 expression in Src-/- versus +/- cells, glucuronidation of DHT versus its Andro metabolite indicates Src inhibits both by 50 %, which necessarily elevates anti-apoptotic DHT levels concomitantly. Exchanges of IYG in wt-UGT-2B15(IYG) and TYS in wt-UGT-2B17(TYS) at positions 98-100 followed by expression in COS-1 and PC3 cells enabled mt-UGT-2B17(IYG) to generated 4 to 10-fold greater in-cellulo caspases 8/3 activations over wt-UGT-2B15(IYG), while mt-UGT-2B15(TYS) suppressed activation of caspases 8/3 over 50% of wt-UGT-2B15 (IYG) levels. Treatment of LNCap cells that contain endogenous UGT-2B15/UGT-2B17 with Src inhibitor, curcumin, enabled a cycle of apoptosis and recovery within 16 h. Combined, evidence indicates the triple-phosphorylated TYS creates a signaling site involving Src and PKCepsilon that is anti-apoptottic, while the pro-apoptotic Src-specific binding/phosphorylation site, IYG, in UGT-2B15 at position 98-100 is blocked by Src. SIGNIFICANCE- This report provides evidence human prostate luminal-cell distributed DHT-metabolizing UGT-2B15 uses programmed phosphorylation-based signaling to carry out luminal-cell specific apoptosis. Contrariwise, basal-cell-distributed UGT-2B17 -- contained within a Basal-cell Compartment that houses intermediate stem cells with both basal- and luminal cell-surface cytokeratin markers-- utilizes programmed anti-apoptotic signaling to protect this population of cells. For the first time, we have identified the luminal-cell specific UGT-2B15 as the pro-apoptotic agent that removes challenged luminal cells before transformation ensues. Moreover we have identified basal-cell specific UGT-2B17 as the anti-apoptotic agent that protects intermediate stem cells containing both basal- and luminal- cell surface markers that replace the departed luminal cell and gives rise to a new basal cell. These findings indicate UGT-2B15 controls luminal transformation and that UGT-2B17 controls continuity of the prostate. Endogenously expressed UGT-2B15/-2B17 in LNCap cells show a cycle of apoptosis and recovery within 16 h after treatment with Src-inhibitor, curcumin. Evidence indicates the triple-phosphorylated TYS creates an anti-apoptotic Src / PKCepsilon signaling site, while pro-apoptotic Src-specific IYG signaling site in UGT-2B15 is blocked by Src phosphorylation. Hence, UGT-2B15 and -2B17 appear to be unique clinical markers. Related manuscript:Identification of a Pro-apoptotic and Anti-apoptotic Controlling Sequence in Human Prostate-distributed DHT-metabolizing UGT-2B15 and UGT-2B17 Respectively Submitted to: Proceedings of the National Academy of Science on 09-2014. In order to establish an in-vivo mammary-gland model that prevents depurination by 4-OH- catecholestrogens associated with initiation of carcinogenesis, we pursued studies to identify mouse homologs of highly effective human UDP-glucuronosyltransferase-2B7. By sequence analysis, we identified mouse Ugt-2b34 and -2b36 homologs that avidly metabolize test-agent 4-OH estrone with 2b35 expressing trivial activity. Unlike low 14M-Km 2B7, 2b34 and 2b36 metabolized 4-OH estrone with 90- and 430-M Km, respectively. Unexpectedly, the isozymes are distributed primarily in male hormone-responsive tissues, whereas 2B7 is found primarily in female hormone-responsive tissues. Also, we found 2b34 metabolizes the non-classical-estrogenic dihydrotestorerone-metabolite, androstane--diol, of DHT at a rate greater than for DHT, which is not known to be estrogenic. Notably, 2B7 essentially failed to metabolize xeno-estrogens; alternatively, 2b34 and 2b36 metabolized BPA and DES at superior rates. Finally, real-time PCR-based analysis of estrogen receptor alpha (ESR1) gene knockout in mouse prostate showed 50% and 63% decreases in Ugt2b34 and Ugt2b36 mRNA, respectively, compared to controls. Contrariwise, estrogen receptor beta (ESR2) k.o. revealed a 2.7/3.3-fold increase in 2b34 and 2b36 mRNA, respectively, in prostate. ESR1 k.o. completely suppressed 2b34 and 2b36 mammary-gland messages; ESR2 k.o. caused a 12-fold increase in 2b34 message without affecting 2b36. Hence it appears males benefit from both Ugts, while females benefit from only one Ugt, according to tissue-distribution studies. Findings for Ugt-2b34 and Ugt-2b36 suggest the two mouse isozymes are intrinsically programmed to protect against a more complex environment than that by human high-activity UGT2B7 and low-activity UGT2B4 and their known distribution patterns. Related manuscript:Prostate-distributed Ugt2b34 and Ugt2b36 Control Estrogenic Metabolites Derived from both Estradiol and DHT in Mice, A. Raychoudhuri, S. Jana, M. Basu, N. K. Basu, and I. S. Owens. Bioscience report (In Press)
