Cytochrome P450c17 catalyzes both 17-hydroxylation and 17,20 oxidative cleavage of C21 steroids, in both human adrenal glands and gonads. While adrenal 17-hydroxylase activity remains fairly constant after birth, adrenal lyase activity, reflected by serum dehydroepiandrosterone sulfate, rises during adrenarche at about age 8 and then declines progressively after age 50. We hypothesize that a post-translational modification of P450c17 regulates its lyase activity. Recently, our laboratory showed that P450c17 is phosphorylated in NCI-H295 cells and that treatment of human fetal adrenal microsomes with alkaline phosphatase abolishes lyase activity without affecting 17-hydroxylation. These data suggest that phosphorylation/dephosphorylation regulates the lyase activity of P450c17. To study the structural and functional consequences of this process in detail, we must first determine the sites and extent of P450c17 phosphorylation. We propose to electrophorese solubilized human adrenal microsomal proteins, with and without alkaline phosphatase treatment, through SDS-polyacrylamide gels. P450c17, identified by size and b Western blotting, willbe excised and cleaved into peptides with trypsin, CNBr, iodosobenzoic acid, etc. The peptide fragments will be characterized by LC/MS or other mass spectrometry methods. Phosphorylated peptides will be identified as additional [M+(PO4)n]+ peaks that are absent in the dephosphorylated sample. Corrected mass rations of M+ to [M+(PO4)n]+ peaks in the native sample will yield the extent of phosphorylation. Results will direct site-directed mutagenesis experiments to systematically abolish phosphorylation sites and to study the functional consequences of such mutations. We will conduct enzymatic experiments to determine the precise step(s) in the P450 catalytic cycle altered by the key phosphorylation events. Finally, we will incorporate results into a computer graphics model of P450c17, constructed by assembling consensus core structural units found in bacterial P450s, to understand the structural basis for the changes in activity regulated by phosphorylation. These data will enable us to better understand and treat clinical problems involving dysregulation and inhibition of androgen biosynthesis, such as the polycystic ovary syndrome and prostatic hyperplasia or cancer.
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