Human cytochrome p450c17: single step purification and phosphorylation of serine 258 by protein kinase a

Abstract

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.

Figure 1

Construction, expression, and purification of recombinant human P450c17. A, Diagram of the modified form of human P450c17 expressed by pCWH17mod(G₃H₆); single letter amino acid codes are used to indicate the relevant sequences. The eight N-terminal residues are modified as described (11), the residues from Leu9 to Thr508 are those of wild-type P450c17 (4,5), and the Gly3His6 tag precedes the stop codon. B, Flow chart of purification of recombinant human P450c17 from bacteria. C, Purification of recombinant human P450c17 from bacteria. Dithionite-reduced carbon monoxide-difference spectra of pCWH17mod(G₃H₆) is shown in the left panel. The specific content is 9.9 pmol/μg protein. In the right panel, samples from various purification steps were analyzed by 12% SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, Cell lysate (∼50 μg protein); lane 2, supernatant from ultracentrifugation (∼50 μg); lane 3, membrane fraction (pellet from ultracentrifugation) (∼25 μg); lane 4, solubilized membrane fraction (∼10 μg); lane 5, flow-through from the Ni-NTA agarose column (∼10 μg); lane 6, purified recombinant human P450c17 (10 μg). The migration of molecular weight markers (kilodaltons) is shown on the left. D, 17α-Hydroxylase and 17,20 lyase activities of the bacterially expressed recombinant human P450c17. Left panel, Radiolabeled progesterone (prog) was used as the substrate for the hydroxylase assays (lanes 1 and 2), and 17OH-preg was used as the substrate for the lyase assays (both at 1 μm). The hydroxylase assay yields 17OHP (17OH-prog) and a small amount of 16OH-progesterone (16OH-prog); the lyase reaction yields dehydroepiandrosterone (DHEA). Some minor contaminant in the substrate steroids are not metabolized. The assays were done in duplicates in lanes 1 and 2 for the hydroxylase reaction and lanes 3 and 4 for the lyase reaction. In lane 5, 10 pmol cytochrome b5 were added to the lyase reaction. Right panel, The activities of pCWH17mod(G₃H₆) were quantitated for the hydroxylase assay (lane 1), lyase assay in the absence of cytochrome b5 (lane 2), and 17,20 lyase activity in the presence of 10 pmol cytochrome b5 (lane 3); data are mean ± sd.

Figure 2

PKA phosphorylates Ser258. In each panel, the amount of P450c17 is indicated by staining with Coomassie brilliant blue (CBB), and phosphorylation is assessed by autoradiography of incorporated ³²P. A, S258A is not phosphorylated. Wild-type (wt) and S258A human P450c17 (3 μg each) were treated without (−) and with (+) 0.5 U PKA catalytic subunit (one time) or with 1.0 U PKA catalytic subunit (two times) and 20 μm ATP (1μCi γ-³²P-ATP) for 30 min at 30 C. B, S256R increases phosphorylation. S256R and wild-type (wt) P450c17 (5 μg each) were incubated with 0.5U PKA catalytic subunit for 10 min at 30 C. The percentages of ³²P incorporated (picomoles ³²P per picomole P450c17) of each sample are indicated below the figure. C, S256R does not promote phosphorylation of S258A. S256R mutant (1 μg) or S256R/S258A double mutant (2 μg) was treated with 0.5 U PKA catalytic subunit for 30 min at 30 C. D, D257A promotes phosphorylation. Wild-type human P450c17, S256R mutant, and S256R/D257A double mutant (0.75 μg each) were treated with 0.5 U PKA catalytic subunit for 10 min at 30 C. The percentages of ³²P incorporation are indicated below the figure, as in B.

Figure 3

Effect of S258 phosphorylation on catalysis by P450c17. A, The S256R mutant (4 μg) was phosphorylated by 0.5 U PKA catalytic subunit with 20 μm ATP (1 μCi γ-³²P-ATP) for the indicated times and analyzed by SDS-PAGE. Staining with Coomassie brilliant blue (CBB) and autoradiography of incorporated ³²P are shown. B, Unphosphorylated and PKA-phosphorylated P450c17 wild-type (left panel) and S256R mutant (right panel) (10 pmol each) were assayed for 17,20 lyase activity with 1 μm ³H-17OH-preg. The lane numbers identify corresponding experiments in each panel. Each form of P450c17 was preincubated in the presence or absence of PKA for 30 min at 30 C followed by lyase assay for 2 h in the absence or presence of 10 pmol cytochrome b5 (cyt b5) in lanes 3 and 6. The experiment was done in duplicates for lanes 1–2 and 4–5 for the unphosphorylated and PKA-phosphorylated forms of P450c17, respectively. DHEA, Dehydroepiandrosterone. C, The S256R mutant was preincubated in the absence or presence of PKA for the indicated times followed by 2 h incubation with steroids for the 17,20 lyase assay in the absence (open bars) or presence (closed bars) of cytochrome b5. The data are means from duplicate experiments.