Human cytochrome p450c17: single step purification and phosphorylation of serine 258 by protein kinase a.
Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity is cAMP-dependent serine/threonine phosphorylation of P450c17. Identifying the responsible kinase(s) and the P450c17 residues that undergo phosphorylation has been challenging, partly because of difficulties in preparing biochemically useful amounts of pure, catalytically active P450c17. We describe a modified strategy for preparing P450c17 in which the traditional carboxy-terminal 4xHis tag is replaced by 3xGly6xHis. This construct permits more rotational freedom of the protein when bound to the nickel affinity column, reducing steric associations between the protein and the column, and permitting a single-step chromatographic purification to apparent homogeneity. Using this vector, we explored P450c17 phosphorylation by mutagenesis of Ser and/or Thr residues to Asp or Glu to mimic the approximate size and charge of phospho-Ser or phospho-Thr. This strategy did not identify Ser and/or Thr site(s) that increase the ratio of lyase to hydroxylase activity, suggesting that the regulatory phosphorylation strategy of human P450c17 is very complicated. Although previous work has excluded protein kinase A (PKA) as the responsible kinase, the cAMP-inducible nature of the phosphorylation-associated increase in lyase activity suggests that PKA may play a role, possibly as a priming kinase. Using our novel vector and a series of mutations, we identified the P450c17 site phosphorylated by PKA as Ser258.
Professor of Pediatrics and Chief of Endocrinology, HSE 1427, University of California, San Francisco, San Francisco, California 94143-0978, USA.
Yue-Hao
YH
Wang
Meng Kian
MK
Tee
Walter L
WL
Miller
D001665:Binding Sites / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D012694:Serine; D013254:Steroid 17-alpha-Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases