Souter Irene, Munir Iqbal, Mallick Parag, Weitsman Stacy R, Geller David H, Magoffin Denis A
Department of Obstetrics and Gynecology, Cedars-Sinai Burns and Allen Research Institute, David Geffen School of Medicine, UCLA, Los Angeles, California, USA.
Fertil Steril. 2006 Apr;85 Suppl 1:1290-9. doi: 10.1016/j.fertnstert.2005.12.011.
To investigate the role of serine-threonine phosphorylation on the activity of human P450c17.
In vitro study.
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.
研究丝氨酸 - 苏氨酸磷酸化对人P450c17活性的作用。
体外研究。
学术基础研究实验室。
无。
构建在蛋白质C末端或N末端带有FLAG标签的P450c17表达载体。对人C末端FLAG标签的P450c17染色体DNA进行定点诱变。将丝氨酸258和苏氨酸260分别突变为丙氨酸和天冬氨酸。在COS - 7细胞中表达突变型P450c17，并测量其酶活性。
人P450c17的17α - 羟化酶和C（17 - 20）裂解酶活性。
C末端FLAG标签的P450c17与野生型P450c17功能无明显差异。突变体S258A、S258D和T260D的17α - 羟化酶和C（17 - 20）裂解酶活性明显低于野生型。
在P450c17蛋白的C末端添加表位标签不会干扰其活性，将是从培养细胞中分离人P450c17蛋白的有用工具。丝氨酸258而非苏氨酸260的磷酸化可能通过与必需的氧化还原伙伴相互作用，作为两种酶活性的生理调节剂。
