Objective: To investigate the role of serine-threonine phosphorylation on the activity of human P450c17.
Design: In vitro study.
Setting: Academic basic research laboratory.
Intervention(s): P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured.
Main outcome measure(s): 17alpha-Hydroxylase and C(17-20) lyase activities of human P450c17.
Result(s): C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17alpha-hydroxylase and C(17-20) lyase activities than the wild type.
Conclusion(s): Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.