title: Mutagenesis of putative serine-threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity.
abstract: 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.
authors.affiliation: Department of Obstetrics and Gynecology, Cedars-Sinai Burns and Allen Research Institute, David Geffen School of Medicine, UCLA, Los Angeles, California, USA.
authors.forename: Irene
authors.initials: I
authors.lastname: Souter
authors.forename: Iqbal
authors.initials: I
authors.lastname: Munir
authors.forename: Parag
authors.initials: P
authors.lastname: Mallick
authors.forename: Stacy R
authors.initials: SR
authors.lastname: Weitsman
authors.forename: David H
authors.initials: DH
authors.lastname: Geller
authors.forename: Denis A
authors.initials: DA
authors.lastname: Magoffin
mesh_terms: 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*
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D013254:Steroid 17-alpha-Hydroxylase