Mutagenesis of putative serine-threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity.
OBJECTIVE
To investigate the role of serine-threonine phosphorylation on the activity of human P450c17.


METHODS
In vitro study.


METHODS
Academic basic research laboratory.


METHODS
None.


METHODS
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.


METHODS
17alpha-Hydroxylase and C(17-20) lyase activities of human P450c17.


RESULTS
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.


CONCLUSIONS
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.
Souter
Irene
I
Department of Obstetrics and Gynecology, Cedars-Sinai Burns and Allen Research Institute, David Geffen School of Medicine, UCLA, Los Angeles, California, USA.
Munir
Iqbal
I
Mallick
Parag
P
Weitsman
Stacy R
SR
Geller
David H
DH
Magoffin
Denis A
DA
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D004789:Enzyme Activation; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D011994:Recombinant Proteins; D013254:Steroid 17-alpha-Hydroxylase