对人细胞色素P450c17的Arg255附近假定的丝氨酸-苏氨酸磷酸化位点进行诱变，并不会选择性地促进其17,20-裂解酶活性。

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

作者信息

Souter Irene, Munir Iqbal, Mallick Parag, Weitsman Stacy R, Geller David H, Magoffin Denis A

机构信息

Department of Obstetrics and Gynecology, Cedars-Sinai Burns and Allen Research Institute, David Geffen School of Medicine, UCLA, Los Angeles, California, USA.

出版信息

Fertil Steril. 2006 Apr;85 Suppl 1:1290-9. doi: 10.1016/j.fertnstert.2005.12.011.

DOI:10.1016/j.fertnstert.2005.12.011

PMID:16616104

Abstract

OBJECTIVE

To investigate the role of serine-threonine phosphorylation on the activity of human P450c17.

DESIGN

In vitro study.

SETTING

Academic basic research laboratory.

PATIENT(S): None.

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.

摘要