Phosphorylation of Human Cytochrome P450c17 by p38α Selectively Increases 17,20 Lyase Activity and Androgen Biosynthesis

Cytochrome P450c17, a steroidogenic enzyme encoded by the CYP17A1 gene, catalyzes the steroid 17 alpha-hydroxylation needed for glucocorticoid synthesis, which may or may not be followed by 17,20 lyase activity needed for sex steroid synthesis. Whether or not P450c17 catalyzes 17,20 lyase activity is determined by three post-translational mechanisms influencing availability of reducing equivalents donated by P450 oxidoreductase (POR). These are increased amounts of POR, the allosteric action of cytochrome b(5) to promote POR-P450c17 interaction, and Ser/Thr phosphorylation of P450c17, which also appears to promote POR-P450c17 interaction. The kinase(s) that phosphorylates P450c17 is unknown. In a series of kinase inhibition experiments, the pyridinyl imidazole drugs SB202190 and SB203580 inhibited 17,20 lyase but not 17 alpha-hydroxylase activity in human adrenocortical HCI-H295A cells, suggesting an action on p38 alpha or p38 beta. Co-transfection of non-steroidogenic COS-1 cells with P450c17 and p38 expression vectors showed that p38 alpha, but not p38 beta, conferred 17,20 lyase activity on P450c17. Antiserum to P450c17 co-immunoprecipitated P450c17 and both p38 isoforms; however, knockdown of p38 alpha, but not knockdown of p38 beta, inhibited 17,20 lyase activity in NCI-H295A cells. Bacterially expressed human P450c17 was phosphorylated by p38 alpha in vitro at a non-canonical site, conferring increased 17,20 lyase activity. This phosphorylation increased the maximum velocity, but not the Michaelis constant, of the 17,20 lyase reaction. p38 alpha phosphorylates P450c17 in a fashion that confers increased 17,20 lyase activity, implying that the production of adrenal androgens (adrenarche) is a regulated event.