Cytochrome P450c17 (P450c17) is the single
enzyme that catalyzes
steroid 17alpha-hydroxylase and
17,20 lyase activities and hence is the crucial decision-making step that determines the class of
steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events.
Serine phosphorylation of P450c17 increases
17,20 lyase activity by increasing the
enzyme's affinity for its redox partner, P450
oxidoreductase. We searched for the relevant
kinase(s) that phosphorylates P450c17 by microarray studies and by testing of
kinase inhibitors. Microarrays show that 145 of the 278 known
serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8-Br-cAMP. Key components of the ERK1/2 and
MAPK/ERK kinase (
MEK)1/2 pathways, which have been implicated in the
insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various
kinase inhibitors that probe the
protein kinase A/
phosphatidylinositol 3-kinase/Akt pathway and the
calcium/
calmodulin/
MAPK kinase pathway had no effect on the ratio of
17,20 lyase activity to 17alpha-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the Rho-associated, coiled-coil containing
protein kinase (ROCK)/Rho pathway suppressed
17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect
17,20 lyase activity. We conclude that members of the ROCK/Rho pathway act upstream from the
kinase that phosphorylates P450c17 in a fashion that augments
17,20 lyase activity, possibly acting to catalyze a priming phosphorylation.