br Phosphorylation of P c Initial
Phosphorylation of P450c17 – Initial studies In addition to the molar ratio of POR to P450c17 and the allosteric action of cytochrome b5, a third factor that governs 17,20 lyase activity is the serine/threonine (Ser/Thr) phosphorylation of P450c17 itself. In a search for post-translational factors that might influence 17,20 lyase activity, we showed that short-term incubation of human adrenal NCI-H295 cells with 32P orthophosphate results in 32P incorporation into P450c17; this was seen in the absence of cAMP but was increased three-fold in 200 µM 8Br-cAMP (Zhang et al., 1995). Similarly, cotransfection with a vector expressing the catalytic subunit of protein kinase A increased incorporation of phosphate two-fold, whereas phorbol ester had no effect, suggesting that the phosphorylation of P450c17 was catalyzed by a cAMP-dependent kinase. Amino 5-moUTP fingerprinting showed that the phosphorylation was in serine and threonine, but not in tyrosine residues. Human fetal adrenal microsomes had robust 17α-hydroxylase and 17,20 lyase activity, but when microsomal proteins were dephosphorylated with alkaline phosphatase, the microsomes lost 17,20 lyase activity, but not 17α-hydroxylase activity, with kinetics that were strictly dependent on the length of time the microsomes were incubated with the phosphatase. By contrast, alkaline phosphatase had no effect on POR activity, hence the loss of 17,20 lyase activity was not due to proteolysis or disruption of the microsomes, and the persistence of 17α-hydroxylase activity showed that the phosphatase had not dephosphorylated NADPH (Fig. 3). Similarly, the treatment with alkaline phosphatase did not alter the substrate-induced difference spectrum of total microsomal P450 incubated with either pregnenolone or 17-Preg, showing that the active site of the enzyme had not been affected. Thus dephosphorylation did not degrade P450c17, but diminished 17,20 lyase activity without altering the substrate binding (Zhang et al., 1995). Studies in other laboratories have confirmed that P450c17 phosphorylation increases 17,20 lyase activity (Biason-Lauber et al, 2000, Kempna et al, 2010). When a protein is activated by phosphorylation there is generally an equilibrium between phosphorylation by the kinase and dephosphorylation by a phosphatase (reviewed by Virshup, 2000). Treating NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin or cantharidin increased 17,20-lyase activity four-fold, suggesting that protein phosphatases 2A (PP2A) and/or PP4 might be involved (Pandey et al., 2003). Neither PP2A nor PP4 affected 17α-hydroxylase activity, but PP2A and not PP4 inhibited 17,20 lyase activity in microsomes from NCI-H295A cells. PP2A co-immunoprecipitated with P450c17, and suppression of PP2A, but not PP4 by siRNA increased 17,20 lyase activity. We found that an endogenous inhibitor of PP2A called phosphoprotein SET is found in NCI-H295A cells; exogenously added SET inhibited PP2A (but not PP4), and increased 17,20 lyase activity in NCI-H295A microsomes. Thus PP2A is the principal phosphatase regulating P450c17 phosphorylation, and PP2A and SET are post-translational factors regulating 17,20 lyase activity and androgen synthesis (Pandey et al., 2003). Because both serine/threonine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, we sought to determine whether these two post-translational mechanisms interact (Pandey and Miller, 2005). A >85% knockdown of cytochrome b5 in NCI-H295A cells with siRNA had no effect on 17α-hydroxylase activity but reduced 17,20 lyase activity by 30%. Treatment of NCI-H295A cells with okadaic acid, which inhibited PP2A and increased P450c17 phosphorylation, could compensate for the knockdown of cytochrome b5. Bacterially-expressed P450c17 could be phosphorylated in vitro by a kinase-enriched fraction from the cytoplasm of NCI-H295A cells; bacterially expressed P450c17 required either this in vitro phosphorylation or the presence of cytochrome b5 to catalyze 17,20 lyase activity. When cytochrome b5 was added to phosphorylated P450c17, the effect on 17,20 lyase activity was not additive; either maximal Ser/Thr phosphorylation or maximal concentrations of cytochrome b5 maximized 17,20 lyase activity. Thus the actions of cytochrome b5 and of P450c17 phosphorylation to enhance 17,20 lyase activity act independently of each other, probably by increasing the interaction between P450c17 and POR (Pandey and Miller, 2005).