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

Mitra PS, Basu NK, Basu M, Chakraborty S, Saha T, Owens IS - J. Biol. Chem. (2010)

Bottom Line: 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-OHE(1) glucuronidation reaching 60% maximum while simultaneously increasing E(2) metabolism linearly.Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE(1), but not E(2), 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.

View Article: PubMed Central - PubMed

Affiliation: Section on Genetic Disorders of Drug Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1830, USA.

ABSTRACT
Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE(1)) with barely detectable 17β-estradiol (E(2)) 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 and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [(33)P]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-OHE(1) at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E(2) 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-OHE(1) glucuronidation reaching 60% maximum while simultaneously increasing E(2) metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E(2) metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF(-/-) is more competent at metabolizing E(2) 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-E(1)-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE(1), but not E(2), 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.

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Effect of 2B7 transfection on 4-OHE1-damaged DNA in comet tails. A, depiction of 2B7 protection against 4-OHE1-based depurination. B, quantitation of protection against 4-OHE1-dependent depurination of COS-1 cells. The cells were treated with vehicle or 100 μm 4-OHE1 for 6 h, processed, and stained before evaluation of damaged DNA using Comet Score v1.5 software as described under “Experimental Procedures.” The results for 200 cells/group were randomly and cumulatively evaluated from three experiments, using two different parameters as shown: percentage of damaged DNA in tail and tail moment (arbitrary units). Student's t test generated a p value of 0.001.
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Figure 6: Effect of 2B7 transfection on 4-OHE1-damaged DNA in comet tails. A, depiction of 2B7 protection against 4-OHE1-based depurination. B, quantitation of protection against 4-OHE1-dependent depurination of COS-1 cells. The cells were treated with vehicle or 100 μm 4-OHE1 for 6 h, processed, and stained before evaluation of damaged DNA using Comet Score v1.5 software as described under “Experimental Procedures.” The results for 200 cells/group were randomly and cumulatively evaluated from three experiments, using two different parameters as shown: percentage of damaged DNA in tail and tail moment (arbitrary units). Student's t test generated a p value of 0.001.

Mentions: Whereas we have demonstrated that Src-supported phosphorylation of 2B7 in COS-1 cells detoxifies 4-OHE1 between 10 and 30% the rate 2B7 expressed in SYF−/−cells, it was of interest to determine whether that level of activity provides measurable protection against 4-OHE1 toxicity. Hence, we adapted the comet assay to score depurination following 4-OHE1 treatment as described under “Experimental Procedures” (26–28). To capture images to demonstrate DNA damage, we first treated nontransfected and 2B7-transfected COS-1 cells with sufficient 4-OHE1, to cause nearly all DNA to redistribute from the head into the comet tail due to damage revealed by SYBR green staining (Fig. 6A, left bottom panel) versus no major DNA redistribution following 4-OHE1 treatment of 2B7-transfected cells (Fig. 6A, right bottom panel). Also, results showed that Me2SO (vehicle) treatment of either nontransfected (Fig. 6A, top left panel) or 2B7-transfected cells (Fig. 6A, top right panel) caused no major change in DNA shift from the head.


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

Mitra PS, Basu NK, Basu M, Chakraborty S, Saha T, Owens IS - J. Biol. Chem. (2010)

Effect of 2B7 transfection on 4-OHE1-damaged DNA in comet tails. A, depiction of 2B7 protection against 4-OHE1-based depurination. B, quantitation of protection against 4-OHE1-dependent depurination of COS-1 cells. The cells were treated with vehicle or 100 μm 4-OHE1 for 6 h, processed, and stained before evaluation of damaged DNA using Comet Score v1.5 software as described under “Experimental Procedures.” The results for 200 cells/group were randomly and cumulatively evaluated from three experiments, using two different parameters as shown: percentage of damaged DNA in tail and tail moment (arbitrary units). Student's t test generated a p value of 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3020772&req=5

Figure 6: Effect of 2B7 transfection on 4-OHE1-damaged DNA in comet tails. A, depiction of 2B7 protection against 4-OHE1-based depurination. B, quantitation of protection against 4-OHE1-dependent depurination of COS-1 cells. The cells were treated with vehicle or 100 μm 4-OHE1 for 6 h, processed, and stained before evaluation of damaged DNA using Comet Score v1.5 software as described under “Experimental Procedures.” The results for 200 cells/group were randomly and cumulatively evaluated from three experiments, using two different parameters as shown: percentage of damaged DNA in tail and tail moment (arbitrary units). Student's t test generated a p value of 0.001.
Mentions: Whereas we have demonstrated that Src-supported phosphorylation of 2B7 in COS-1 cells detoxifies 4-OHE1 between 10 and 30% the rate 2B7 expressed in SYF−/−cells, it was of interest to determine whether that level of activity provides measurable protection against 4-OHE1 toxicity. Hence, we adapted the comet assay to score depurination following 4-OHE1 treatment as described under “Experimental Procedures” (26–28). To capture images to demonstrate DNA damage, we first treated nontransfected and 2B7-transfected COS-1 cells with sufficient 4-OHE1, to cause nearly all DNA to redistribute from the head into the comet tail due to damage revealed by SYBR green staining (Fig. 6A, left bottom panel) versus no major DNA redistribution following 4-OHE1 treatment of 2B7-transfected cells (Fig. 6A, right bottom panel). Also, results showed that Me2SO (vehicle) treatment of either nontransfected (Fig. 6A, top left panel) or 2B7-transfected cells (Fig. 6A, top right panel) caused no major change in DNA shift from the head.

Bottom Line: 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-OHE(1) glucuronidation reaching 60% maximum while simultaneously increasing E(2) metabolism linearly.Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE(1), but not E(2), 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.

View Article: PubMed Central - PubMed

Affiliation: Section on Genetic Disorders of Drug Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1830, USA.

ABSTRACT
Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE(1)) with barely detectable 17β-estradiol (E(2)) 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 and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [(33)P]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-OHE(1) at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E(2) 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-OHE(1) glucuronidation reaching 60% maximum while simultaneously increasing E(2) metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E(2) metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF(-/-) is more competent at metabolizing E(2) 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-E(1)-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE(1), but not E(2), 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.

Show MeSH
Related in: MedlinePlus