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

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

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / chemistry*
  • Amino Acids / genetics
  • Amino Acids / metabolism*
  • Animals
  • Binding Sites
  • COS Cells
  • Chlorocebus aethiops
  • Computer Simulation
  • Enzyme Activation
  • Humans
  • Models, Chemical*
  • Models, Molecular*
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Steroid 17-alpha-Hydroxylase / chemistry*
  • Steroid 17-alpha-Hydroxylase / genetics
  • Steroid 17-alpha-Hydroxylase / metabolism*

Substances

  • Amino Acids
  • Recombinant Proteins
  • Steroid 17-alpha-Hydroxylase