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Endocrinology, doi:10.1210/en.2007-1527
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Endocrinology Vol. 149, No. 5 2667-2677
Copyright © 2008 by The Endocrine Society

Pathways Leading to Phosphorylation of P450c17 and to the Posttranslational Regulation of Androgen Biosynthesis

Meng Kian Tee, Qing Dong and Walter L. Miller

Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco (UCSF), San Francisco, California 94143

Address all correspondence and requests for reprints to: Professor Walter L. Miller, Department of Pediatrics, HSE 1427, University of California, San Francisco, San Francisco, California 94143-0978. E-mail: wlmlab{at}ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytochrome P450c17 (P450c17) is the single enzyme that catalyzes steroid 17{alpha}-hydroxylase and 17,20 lyase activities and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme’s affinity for its redox partner, P450 oxidoreductase. We searched for the relevant kinase(s) that phosphorylates P450c17 by microarray studies and by testing of kinase inhibitors. Microarrays show that 145 of the 278 known serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8-Br-cAMP. Key components of the ERK1/2 and MAPK/ERK kinase (MEK)1/2 pathways, which have been implicated in the insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various kinase inhibitors that probe the protein kinase A/phosphatidylinositol 3-kinase/Akt pathway and the calcium/calmodulin/MAPK kinase pathway had no effect on the ratio of 17,20 lyase activity to 17{alpha}-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the Rho-associated, coiled-coil containing protein kinase (ROCK)/Rho pathway suppressed 17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect 17,20 lyase activity. We conclude that members of the ROCK/Rho pathway act upstream from the kinase that phosphorylates P450c17 in a fashion that augments 17,20 lyase activity, possibly acting to catalyze a priming phosphorylation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE POLYCYSTIC OVARY syndrome (PCOS), which affects about 6% of women of reproductive age (1), is primarily characterized by hyperandrogenism, hyperandrogenemia, infrequent menses with oligo/anovulation, and disordered gonadotropin secretion and is frequently associated with insulin resistance and the metabolic syndrome (2). Although most PCOS investigators focus on the ovary, clinical data indicate that, in many PCOS women, the hyperandrogenemia is of both ovarian and adrenal origin; when adrenal steroidogenesis is suppressed with dexamethasone, hyperandrogenism persists, indicating ovarian production (3, 4), and when ovarian steroidogenesis is suppressed by suppressing gonadotropins, the hyperandrogenism persists, indicating an adrenal source (5, 6, 7). Because the pathways of androgen synthesis in the adrenal and ovary employ the same enzymes (8) and because the hyperandrogenemia appears to be the primary event, we have focused on cellular mechanisms regulating androgen biosynthesis.

The synthesis of all steroid hormones begins with the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc (8). The level of expression of P450scc determines cellular steroidogenic capacity (9), and the expression of steroidogenic acute regulatory protein (StAR) regulates short-term levels of steroidogenesis (10, 11), so that these two factors constitute the quantitative regulators of steroidogenesis. By contrast, the qualitative regulation, determining the class of steroid produced by a cell, is determined by microsomal P450c17 (12), the single enzyme that catalyzes both 17{alpha}-hydroxylation and 17,20 lyase activity (13, 14, 15, 16). The adrenal zona glomerulosa does not express P450c17 (17) and hence produces 17-deoxysteroids leading to aldosterone. The adrenal zona fasciculata expresses the 17{alpha}-hydroxylase activity but very little of the 17,20 lyase activity of P450c17, and hence produces 21-carbon, 17-hydroxysteroids, leading to cortisol. The adrenal reticularis, testicular Leydig cells, and ovarian theca cells express both the 17{alpha}-hydroxylase and 17,20 lyase activities of P450c17, and hence produce the 19-carbon 17-hydroxy steroid dehydroepiandrosterone (DHEA), the precursor of sex steroids. Thus, the cellular events determining whether P450c17 catalyzes only the 17{alpha}-hydroxylase reaction or both the 17{alpha}-hydroxylase and 17,20 lyase reactions are key components in regulating androgen synthesis.

Human beings and other higher primates undergo adrenarche shortly before the onset of puberty, at which time adrenal secretion of DHEA and DHEA-sulfate (DHEAS) increase about 10-fold while the production of cortisol remains constant (18, 19). Two factors are responsible for this change in the zona reticularis: a decrease in expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) (17, 20, 21) and the acquisition of 17,20 lyase activity by P450c17. The acquisition of 17,20 lyase activity may be mediated by three events, all of which act to increase the efficiency of electron transfer from NADPH via P450 oxidoreductase (POR) to P450c17 (22). First, increasing the molar ratio of POR to P450c17 increases the lyase activity (23, 24); second, cytochrome b5 can act as an allosteric factor to promote the interaction of POR and P450c17 to promote 17,20 lyase activity (25, 26); and third, phosphorylation of P450c17 on serine and threonine residues selectively increases 17,20 lyase activity (27, 28, 29). Although it is likely that more than one of these mechanisms of increasing 17,20 lyase activity may function in adrenarche, we have proposed that the serine phosphorylation of P450c17 may be directly linked with the hyperandrogenism and insulin resistance seen in a subset of patients with PCOS (12, 27, 29).

Insulin acts by binding to extracellular {alpha}-subunits of the insulin receptor (IR), eliciting a conformational change in the intracellular domain of the β-chain (IRβ), which activates the tyrosine kinase domain of IRβ (30). The tyrosine-phosphorylated IRβ then tyrosine phosphorylates and activates the four IR substrate (IRS) proteins (IRS1–4) and possibly other factors (31). The activated IRS proteins then transmit the insulin signal via multiple intracellular pathways, especially those initiated by phosphatidylinositol-3-kinase (PI3K) and by MAPK (32, 33). If IRβ is phosphorylated on serine residues, insulin binding is unaffected but IRβ tyrosine phosphorylation is inhibited, interfering with insulin action (34, 35, 36, 37). Some women with PCOS have increased serine phosphorylation of IRβ (38, 39) or of IRS-1 (40). Because increased serine phosphorylation of IRβ is a mechanism of insulin resistance and serine phosphorylation of P450c17 increases its androgen biosynthetic capacity, we have proposed that a subset of women with an autosomal dominant form of PCOS would have a gain-of-function mutation in a factor common to both pathways (27, 41).

The identification of the relevant factors remains elusive. Because the onset of adrenarche is contemporaneous with the rise in IGF-I, we suggested that IGF-I may be involved in adrenarche and PCOS (27). Both IGF-I and insulin share the PI3K pathway through Akt. One study implicated the PI3K pathway but not the MAPK pathway in insulin signaling in human theca cells (42); others found increased serine phosphorylation of ERK1/2 and MAPK/ERK kinase (MEK)1/2 in muscle of PCOS women, implicating the MAPK pathway (43), but that study did not examine potential effects on androgen biosynthesis. We now report a survey of potential factors leading to the phosphorylation of P450c17, identifying Rho-associated, coiled-coil containing protein kinase (ROCK)1 as a potential factor, providing further evidence linking the hyperandrogenism and insulin resistance of PCOS through a single serine phosphorylation mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Microarrays and data analysis
NCI-H295A adrenocortical cells (44, 45, 46) were grown on 10-cm plates in RPMI 1640 with 2% fetal calf serum, gentamicin, insulin, and selenium. Three nearly confluent plates were incubated in the presence and absence of 1 mM 8-Br-cAMP for 18 h; the cells were washed with PBS and lysed with 1 ml Trizol (Invitrogen, Carlsbad, CA). Total RNA was purified further using RNeasy columns (QIAGEN, Valencia, CA), and 4 µg RNA was used to synthesize double-stranded cDNA using the MessageAmp II-Biotin Enhanced Kit (Ambion, Austin, TX) with oligo-dT primer [5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24] to incorporate a T7 RNA polymerase promoter. Antisense cRNA was labeled with biotin-UTP using T7 RNA polymerase (Ambion), and the cRNA was fragmented to 35–200 nucleotides (nt) at 94 C for 35 min in 40 µl 40 mM Tris-acetate (pH 8.1), 100 mM KOAc, 30 mM MgOAc. The integrities of total RNAs, cRNAs, and fragmented cRNAs were assessed on a Bio-Rad (Hercules, CA) Experion BioAnalyzer. Each sample of fragmented cRNA was initially hybridized with Affymetrix (Santa Clara, CA) Test3 arrays to determine the quality of the labeled probe before hybridization to Affymetrix U133A plus 2.0 arrays. The chips were stained and washed using an Affymetrix GeneChip Fluidics Station 450, and the arrays were scanned using a GCS 3000 scanner with auto-loader in the Genomics Core laboratory. Hybridization intensity signals from the scanned images were analyzed using Affymetrix GeneChip Operating Software (GCOS, version 1.3) to provide qualitative detection calls and quantitative estimates of gene expression levels (signal values). Raw data were exported to Microsoft Excel for analysis. Transcripts displaying a cAMP-induced increased or decreased signal were selected for further analysis only if they had a mean ± 2.0-fold change and were statistically significant (P < 0.05) in three separate experiments; transcripts displaying no signal change relative to controls in at least three experiments were excluded from further analysis.

Kinase inhibitor treatments
COS-1 monkey kidney cells were grown in DMEM H21 with 10% fetal calf serum and gentamicin. NCI-H295A cells or COS-1 cells transfected with the pCDNA3 vector expressing human P450c17 (14) were treated with H-1152 (10 µM), HA-1077 (30 µM), Kn-62 (10 µM), LY294002 (100 µM), ML-9 (20 µM), myristoylated protein kinase A (PKA) inhibitor amide 14–22 (1 µM), rapamycin (1 µM), U0126 (20 µM), or wortmannin (200 µM) for 3.5 h (all inhibitors from EMD Biosciences, San Diego, CA). To assay P450c17 activities, cells were preincubated with 10 µM cyanoketone (a kind gift from Dr. Mary Dallman, UCSF) for 30 min to inhibit 3β-HSD, followed by incubation with labeled steroid for 1 h; 17{alpha}-hydroxylase assays used 5 µM [14C]progesterone (55.4 mCi/mmol; PerkinElmer, Norwalk, CT), and 17,20 lyase assays used 0.8 µM [3H]17-hydroxypregnenolone (60 mCi/mmol; American Radiolabeled Chemicals, St. Louis, MO). Steroids were extracted and separated by thin-layer chromatography (TLC) as described (16) and quantitated by phosphorimaging using Scion Image software (Frederick, MD). Inhibitor experiments were done in triplicate using 12-well plates, except for the experiments with rapamycin and wortmannin, which were done twice.

Transfections
Expression vectors for ROCK1 fused to myc in pCAGmyc were kindly provided by Dr. Shuh Narumiya, Kyoto University, Japan. Wild-type ROCK1 (ROCK1-wt), which contains 1354 amino acids, and two constitutively active mutants lacking the C-terminal autoinhibitory domain, ROCK1-{Delta}1 lacking 274 amino acids and ROCK1-{Delta}3 lacking 627 amino acids, were each fused to myc on their C termini (47). Cells were transfected using Effectene (QIAGEN) according to the manufacturer’s protocol.

Bacterial expression of P450c17 and P450 oxidoreductase
The pCWH17-mod(His)4 expression plasmid containing the cDNA for human P450c17 with amino-terminal modifications that facilitate bacterial expression (48) was transformed into Escherichia coli strain JM109. Ampicillin-resistant colonies were grown at 37 C to A600 0.4, and P450c17 expression was induced with 0.4 mM isopropyl-1-thio-β-D-galactopyranoside at 28 C for 36 h and purified as described (48). Briefly, spheroplasts prepared by treatment with lysozyme were lysed by sonication and centrifuged at 4000 x g for 10 min, and the membrane pellet containing P450c17 was solubilized in 0.7% Triton X-114 (Calbiochem, La Jolla, CA) and centrifuged at 100,000 x g for 30 min. The reddish-brown detergent-rich supernatant fraction containing P450c17 was collected and mixed with Ni-NTA-Sepharose beads, and the beads were washed and eluted with 200 mM histidine. The eluted P450c17 was purified further by hydroxyapatite chromatography to remove histidine and other protein contaminants. Human POR cDNA lacking the codons for its 27 N-terminal residues was expressed, purified, and quantitated as described (49).

In vitro assays of P450c17
Ten picomoles of P450c17 and 20 pmol POR, each expressed in bacteria, were emulsified with 20 µg phosphatidylcholine in 100 mM potassium phosphate, 6 mM potassium acetate, 10 mM MgCl2, 1 mM reduced glutathione, 20% glycerol, 3 U glucose-6-phosphate dehydrogenase, and 0.1 mM glucose-6-phosphate and incubated for 3 h at 37 C with either [14C]progesterone or [3H]17-hydroxypregnenolone in a total volume of 200 µl. For assays of the 17,20 lyase reaction, 10 pmol cytochrome b5 (Invitrogen) were added to the P450c17-POR reaction mixture.

In vitro phosphorylation
Purified, bacterially expressed human P450c17 (1 µg) was incubated with catalytically active recombinant p70S6K (Upstate Biotechnology, Lake Placid, NY), PKA (New England Biolabs, Ipswich, MA), or PKN1 or ROCK1 (Invitrogen) in 20 mM HEPES, 20 mM MgCl2, 200 µM [{gamma}-32P]ATP (6000 Ci/mmol; PerkinElmer) for 20 min at 30 C. P450c17 was captured on Ni-NTA beads, washed 10 times with 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and eluted in SDS-gel loading buffer. Incorporated radioactivity was quantitated by scintillation counting. Bound protein was eluted with SDS sample buffer at 95 C for 2 min, displayed by SDS-PAGE, and analyzed by phosphorimaging using a Storm phosphorimager (Amersham, Piscataway, NJ).

Immunoprecipitation and Western blotting
Cells were lysed in 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1 mM EDTA, 10% glycerol, and protease inhibitors. Cell lysates were precleared with protein A agarose and incubated with antisera to P450c17 (24) 1:2000, c-myc 1:2000 (BD BioSciences), or ROCK1 1:2000 (Invitrogen) at 4 C overnight on a rocking platform. Immunocomplexes were captured onto protein A agarose beads (50 µl of a 50% slurry) (Invitrogen), washed, eluted in SDS sample buffer, subjected to SDS-PAGE, and blotted onto polyvinylidene difluoride membrane. Western blotting was done in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20, 5% skim milk, and 5% horse serum; bands were visualized by the ECL plus reagent (GE HealthCare, Piscataway, NJ) and recorded by phosphorimaging.

In vitro pull-down assay
Catalytically active glutathione S-transferase (GST)-tagged ROCK1 (Invitrogen) was incubated with purified, bacterially expressed His-tagged P450c17 in 20 mM HEPES, 20 mM MgCl2, 200 µM ATP for 20 min at 30 C. Protein complexes were captured onto GST or Ni-NTA beads, washed three times in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, eluted in SDS-gel loading buffer, displayed by SDS-PAGE, and stained by Coomassie Blue R250. Alternatively, recombinant ROCK1 was prebound to GST beads and incubated with soluble, bacterially expressed P450c17, or recombinant P450c17 was bound to Ni-NTA beads and incubated with ROCK1 in 20 mM HEPES, 20 mM MgCl2, 200 µM ATP for 20 min at 30 C. The complexes were washed, eluted, displayed by SDS-PAGE, and stained by Coomassie Blue R250.

RNA interference
The lentilox PLL 3.7 vector (50) was used for ROCK1 and ROCK2 small interfering RNA (siRNA) constructs: ROCK1 sequence 5'-AGGTGATTGGTAGAGGTGCA-3' (nt 239–258) and ROCK2 sequence 5'-AAGGCATCGCAGAAGGTTTAT-3' (nt 783–803). A scrambled sequence, 5'-ACATTGAAGCGAAGAATAA-3', was used as a negative control. To make siRNA constructs to the six cAMP-activated kinases, the following sequences were used: BMP2 inducible kinase (BMP2K), 5'-AAGGTTGCATCAGTGTAAGAC-3' (nt 667–687); G protein-coupled receptor kinase 5 (GRK5), 5'-AAGCCGTGCAAAGAACTCTTT-3' (nt 639–359); tribbles homolog 1 (TRIB1), 5'-AAACCAAGGCCTATGTCTTCT-3' (nt 1040–1060); testis-specific kinase 1 (TESK1), 5'-AAAGGTGTATTTCACCGCGAC-3' (nt 841–861); and MAPK13/p38{delta}, 5'-AATGAGGACTGTGAACTGAAG-3' (nt 573–593). The constructs were cotransfected with packaging vectors into HEK-293T by Effectene (QIAGEN). The supernatants containing lentiviral particles were filtered on 0.45-µm polyvinylidene difluoride membranes and used to infect human adrenal NCI-H295A cells (45, 46) for 2 d, and hydroxylase and lyase assays were done as described for the kinase inhibitor assays.

To confirm the suppression of ROCK1 and ROCK2 expression by siRNA lentiviral infection in NCI-H295A cells, RT-PCR was done using the following primers: ROCK1 sense 5'-AGCCGCCGGGACCCAACTATCGT-3' (nt 192–214) and antisense 5'-CTGTGCCAGCTGCGGCCGCCG-3' (nt 489–469) and ROCK2 sense 5'-CTACAGATGAAGGCAGAAGACTAT-3' (nt 702–725) and antisense 5'-GCATCCAGAGCAAGAACAAC-3' (nt 1060–1041). β-Glucuronidase primers sense 5'-CGGCATTTTGTCGGCTGGGTGTG-3' (nt 303–325) and antisense 5'-GATGTAGGTGGTGGGTGTCGTGTA-3' (nt 710–687) were used for internal control.

In vivo labeling
Phosphoproteins in NCI-H295A cells or in COS-1 cells transfected with a P450c17 expression vector were labeled with [32P]orthophosphate free of carrier and HCl (0.8 mCi/ml; GE HealthCare) for 4 h in phosphate-free DME-H21 with and without dialyzed 10% fetal calf serum. To deplete endogenous phosphate before labeling, cells were rinsed twice and preincubated with phosphate-free DME-H21 for 1 h. Cells were lysed in 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1 mM EDTA, and 10% glycerol and protease inhibitors, and P450c17 was immunoprecipitated and analyzed by SDS-PAGE as described above.

Site-directed mutagenesis
Serines at positions 94, 256, 258, and 273 in pCMV-P450c17 were mutated to alanine to prevent phosphorylation or to aspartic acid and glutamic acid to mimic negatively charged phosphoserine. Mutagenesis was done with the following PCR primer sequences: S94A, AAGGGCAAGGACTTCGCCGGGCGGCCTCAAATG-3'; S94D, 5'-AAGGGCAAGGACTTCGACGGGCGGCCTCAAATG-3'; S94E, 5'-AAGGGCAAGGACTTCGAAGGGCGGCCTCAAATG-3'; S256A, AAGGAGAAATTCCGGGCCGACTCTATCACCAAC-3'; S256D, 5'-AAGGAGAAATTCCGGGACGACTCTATCACCAAC-3'; S256E, 5'-AAGGAGAAATTCCGGGAAGACTCTATCACCAAC-3'; S258A, 5'-AAATTCCGGAGTGACGCCATCACCAACATGCTG-3'; S258D, 5'-AAATTCCGGAGTGACGACATCACCAACATGCTG-3'; S258E, 5'-AAATTCCGGAGTGACGAAATCACCAACATGCTG-3'; S273A, 5'-CAAGCCAAGATGAACGCCGATAATGGCAATGCT, S273D, 5'-CAAGCCAAGATGAACGACGATAATGGCAATGCT-3'; and S273E, 5'-CAAGCCAAGATGAACGAAGATAATGGCAATGCT-3' (the mutated bases are underlined). The double alanine mutant construct S94A + S273A was created by ligation of a 5942-nt BstEII-EcoRI fragment of construct S94A with a 1006-nt BstEII-EcoRI fragment of construct S273A. The constructs were transfected into COS cells, and 17{alpha}-hydroxylase and 17,20 lyase assays were done in the presence or absence of 1 µM H-1152P inhibitor as described above.

Statistical analyses
Statistical analyses were performed using two-tailed unpaired t tests, and significance was accepted for tests where P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P450c17 phosphorylation and 17,20 lyase activity
NCI-H295 cells, derived from a human adrenocortical carcinoma (44), express P450c17, catalyze 17{alpha}-hydroxylase and 17,20 lyase activities (45, 46), and can phosphorylate P450c17 in a fashion that maximizes 17,20 lyase activity (29, 51). Therefore, these cells provide an appropriate environment in which to study the phosphorylation of human P450c17. To examine the potential effects of growth conditions on the phosphorylation of P450c17 in NCI-H295 cells, we incubated NCI-H295A cells with [32P]orthophosphate for 4 h in the presence or absence of serum. Serum deprivation increased 32P incorporation 3.5-fold compared with serum-treated cells, indicating an inhibition of P450c17 phosphorylation by serum (Fig. 1AGo). COS-1 African green monkey kidney cells do not express endogenous P450c17 but can be used to assess 17,20 lyase activity and the phosphorylation status of transfected human P450c17 (27). To determine whether serum deprivation affects P450c17 enzymatic activities, NCI-H295A cells and COS-1 cells transfected with P450c17 were incubated in the presence or absence of serum or insulin with [14C]progesterone to assay 17{alpha}-hydroxylase activity or with [3H]17-hydroxypregnenolone to assay 17,20 lyase activity (Fig. 1BGo). In the presence of serum (lanes 1, 3, and 6), the hydroxylase activity was unaffected, but 17,20 lyase activity was decreased. When insulin was added in the absence of serum, the ratio of 17,20 lyase to 17{alpha}-hydroxylase activities was no different from in the absence of serum alone (Fig. 1CGo). Therefore, P450c17 appears to be phosphorylated by a kinase that is suppressed by serum.


Figure 1
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  		FIG. 1. Serum suppresses P450c17 phosphorylation. A, NCI-H295A cells were labeled with [32P]orthophosphate in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of serum, followed by immunoprecipitation (lanes 1 and 2) or immunoblotting (IB) with P450c17 antibody (lanes 3 and 4). B, NCI-H295A cells (left panel) and COS1 cells transfected with P450c17 (right panel) were assayed for 17{alpha}-hydroxylase and 17,20 lyase activities in the presence or absence of serum and 100 nM insulin for 4 h. The samples in lanes 4 and 7 received insulin without serum. The controls received the complete medium with serum. C, The above experiment was done in triplicate to determine the ratio of 17,20 lyase to 17{alpha}-hydroxylase activity in NCI-H295A cells. Asterisks indicate significant differences from controls: P = 0.00070 and P = 0.00060 for the treatments minus serum/minus insulin and minus serum/plus insulin, respectively. 17-OH preg, 17-Hydroxypregnenolone; 17-OH prog, 17-hydroxyprogesterone.

 
Microarray experiments
Of the 518 kinases in the human kinome, 278 are serine/threonine kinases (52). In an initial attempt to identify kinases that might phosphorylate P450c17, we analyzed microarrays. No commercially available microarray focuses on kinases and related factors, but the U133 plus 2.0 chip features 54,120 probe sets representing 38,573 gene clusters in the UniGene database plus 841 anonymous full-length transcripts and a number of anonymous expressed sequence tags, yielding 47,401 putatively unique transcripts (53). The 518 kinase genes are represented in the UniGene database; hence, this chip provides full kinase coverage. RNA from three independent batches of NCI-H295A cells showed that the mRNAs for P450scc, P450c21, P450c17, StAR, POR, and cytochrome b5 were all readily detected, showing that the chips detected known adrenal mRNAs. Of the 278 known serine/threonine kinases, 145 were detected in NCI-H295A cells (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Among the kinases not found (supplemental Table 2), several kinases that might logically be implicated in a pathway associated with the action of insulin or IGF-I were notable by their absence, including mammalian target of rapamycin (mTOR), MAPK3/ERK1, MAPK1/ERK2, MAP2K1/MEK1, and MAP2K2/MEK2. Thus, ERK1/2 and MEK1/2, which have been implicated in the insulin resistance of PCOS muscle cells (40, 43), cannot be involved in the phosphorylation of human P450c17 in NCI-H295A cells.

Because the activation of androgen synthesis in adrenarche and in PCOS appears to be chronic, we considered that the relevant kinase(s) may be inducible at the transcriptional level. Because cAMP increases the serine phosphorylation of P450c17 (27), we compared triplicate control microarrays with triplicate microarrays of RNA from NCI-H295A cells treated overnight with 1 mM 8-Br-cAMP. As expected, the mRNAs for P450scc, P450c21, P450c17, StAR, and POR were all induced by cAMP, whereas the mRNA for cytochrome b5 was reduced by about half (Table 1Go). Thus, the NCI-H295A cells responded as expected to treatment with 8-Br-cAMP. Using an arbitrary ±2-fold change in expression levels compared with control, a total of 228 genes were inhibited (supplemental Table 3), and 140 genes were activated (supplemental Table 4) by the cAMP treatment. The 228 genes that were inhibited by cAMP included eight serine/threonine kinases, two tyrosine kinases, one serine/threonine phosphatase, one tyrosine phosphatase, and 12 other transcripts associated with kinases or involved in the regulation of kinase pathways (Table 2Go).


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  		TABLE 1. Expression of steroidogenic genes in NCI-H295A cells treated with 8-Br-cAMP

 

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  		TABLE 2. cAMP-mediated suppression of kinases, phosphatases, and other genes that participate in phosphorylation/dephosphorylation pathways

 
Among the 140 genes that were induced by cAMP, six were serine/threonine kinases (BMP2K, GRK5, MAPK13, SNF1LK, TESK1, and TRIB1), one was a dual-specificity phosphatase, three were tyrosine phosphatases, and nine others were either associated with kinases or involved in the regulation of kinase pathways (Table 3Go). Among the genes induced by cAMP, MAPK13, also known as p38{delta}, is the major okadaic acid-responsive p38 MAPK isoform in keratinocytes, and MAPK13 can inactivate ERK1/2 activity directly by forming a p38{delta}-ERK1/2 complex (54). PCK1 has been implicated in type 2 diabetes mellitus, and the lack of PCK1 expression can cause insulin resistance in mice (55). TRIB1 can activate the MAPK kinase pathway (56), which is induced in muscle cells in PCOS women (40). Because pharmacological inhibitors to BMP2K, GRK5, MAPK13, SNF1LK, TESK1, and TRIB1 are not available, we used RNA interference to suppress the expression of these kinases. Lentiviruses expressing siRNAs to BMP2K, GRK5, MAPK13, SNF1LK, TESK1, and TRIB1 were prepared from HEK-293T cells and used to infect subconfluent NCI-H295A cells. We found no difference in 17{alpha}-hydroxlyase and 17,20 lyase activities compared with control cells infected with lentiviruses expressing a scrambled sequence (data not shown).


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  		TABLE 3. cAMP-mediated induction of kinases, phosphatases, and other genes that participate in phosphorylation/dephosphorylation pathways

 
Effect of kinase inhibitors on 17,20 lyase activity
The microarray experiments ruled out potential roles for many kinases but did not provide information about the potential involvement of the PKA/PI3K pathway. Signaling by insulin and by IGF-I is, in part, mediated by the cAMP-responsive PKA/PI3K/Akt pathway, whereas experiments with muscle cells from PCOS women suggest a role for components of the insulin-inducible MAPK pathway (40, 43). To examine the potential roles of this pathway and others not excluded by the microarray work, we examined the effects of various kinase inhibitors on the activities of P450c17. The inhibitors used and their known target kinases are listed in Table 4Go. Eight inhibitors (H89, Kn-62, LY294002, ML-9, myristoylated PKA inhibitor amide 14–22, rapamycin, U0126, and wortmannin) that probe the PKA/PI3K/Akt/mTOR pathway and the calcium-calmodulin kinase (CaMK)/myosin light chain kinase (MLCK)/MEK1/MEK2 pathway had no effect on the ratio of 17,20 lyase to 17{alpha}-hydroxylase activities, suggesting that these widely studied kinase pathways are not key players in the phosphorylation of P450c17 (Table 4Go). However, 30 µM HA-1077, which inhibits a number of kinases, decreased 17,20 lyase activity modestly. Most of the kinases inhibited by HA-1077 were already tested by one or more of the other inhibitors discussed above. To explore the potential roles of other kinases inhibited by HA-1077, we incubated NCI-H295A cells with 10 µM HA-1152, a highly specific inhibitor of ROCK1 and ROCK2 (57); H-1152 suppressed the ratio of 17,20 lyase to 17{alpha}-hydroxylase activity to 68% of control (Table 4Go). A similar inhibition was seen in COS1 cells transfected with a P450c17 expression vector (data not shown). These data suggest that an H-1152-sensitive kinase, possibly ROCK1/2, participates in regulating the 17,20 lyase activity of P450c17.


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  		TABLE 4. Effect of kinase inhibitors of P450c17 on the ratio of 17,20 lyase to 17{alpha} -hydroxylase activities in NCI-H295A cells

 
Overexpression of ROCK1 induces 17,20 lyase activity
To examine the potential effects of ROCK1 on 17,20 lyase activity, COS1 cells were cotransfected with P450c17 and myc-tagged ROCK1-wt or the constitutively active C-terminally deleted ROCK1-{Delta}1 and -{Delta}3 (47). Overexpression of all three forms of ROCK1 increased the ratio of 17,20 lyase to 17{alpha}-hydroxylase activity by about 1.5-fold (Fig. 2Go). Thus, ROCK1 augments 17,20 lyase activity, but it was not clear whether ROCK1 acts directly on P450c17 or acts on another factor.


Figure 2
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  		FIG. 2. ROCK1 overexpression induces 17,20 lyase activity. COS-1 cells transfected with P450c17 alone or with the indicated ROCK1 constructs were incubated with [14C]progesterone (prog) (top panel) or [3H]17-hydroxypregnenolone (17-OH preg) (bottom panel) for 1 h. Steroids were extracted and analyzed by TLC and autoradiography. The data (mean ± SD) from three separate experiments are summarized on the right. Asterisks indicate significant differences from controls: P = 0.043, P = 0.0062, P = 0.0037, and P = 0.0022 for cells transfected with P450c17 and treated with cAMP, cotransfected with P450c17 and p160ROCK1-wt, p160ROCK1-{Delta}1 and p160ROCK1-{Delta}3, respectively.

 
ROCK1 phosphorylation of P450c17 is not sufficient to induce 17,20 lyase activity
To determine whether ROCK1 or other kinases can phosphorylate P450c17 directly, we prepared human P450c17 and POR in bacteria and purified each nearly to homogeneity (Fig. 3AGo). Incubation of the bacterially expressed P450c17 with 200 µM [{gamma}-32P]ATP alone or with the cAMP-dependent kinase p70S6K yielded no phosphorylation of P450c17, but incubation with PKA yielded low-level incorporation, and incubation with ROCK1 yielded robust incorporation of 32P (Fig. 3BGo). Thus, both PKA and ROCK1 can phosphorylate P450c17.


Figure 3
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  		FIG. 3. Recombinant ROCK1 alone can phosphorylate P450c17 in vitro but is not sufficient to induce 17,20 lyase activity. A, POR and His-tagged P450c17 (c17) were expressed in bacteria, purified, and analyzed by SDS-PAGE. B, Purified P450c17 (1 µg) was incubated with catalytically active recombinant p70S6K (S6K), PKA, or ROCK1 in 200 µM [{gamma}-32P]ATP for 20 min at 30 C. P450c17 was captured onto Ni-NTA beads, washed and eluted in SDS-gel loading buffer, and analyzed by SDS-PAGE. C, P450c17 was preincubated with recombinant ROCK1 in 20 mM HEPES, 20 mM MgCl2, 200 µM ATP for 20 min at 30 C. POR (60 pmol), cytochrome b5 (10 pmol), and substrates [14C]progesterone (prog) or [3H]17-hydroxypregnenolone (17-OH preg) were added, and the mixtures were incubated at 37 C for 3 h. Steroids were extracted and analyzed by TLC and autoradiography.

 
To determine whether ROCK1 phosphorylation of P450c17 increases 17,20 lyase activity, we repeated the procedure of Fig. 2BGo with unlabeled ATP and then assayed the 17{alpha}-hydroxylase and 17,20 lyase activities of P450c17 in the presence of bacterially expressed POR, with and without P450c17 phosphorylation by ROCK1 (Fig. 3CGo). The bacterially expressed P450c17 had 17{alpha}-hydroxylase activity (conversion of progesterone to 17-hydroxyprogesterone) that was not affected by ROCK1 (lanes 1 and 2). The capacity of the bacterially expressed P450c17 to catalyze 17,20 lyase activity was shown by its ability to convert 17-hydroxypregnenolone to DHEA in the presence of cytochrome b5 (lane 5); however, phosphorylation of P450c17 by ROCK1 did not increase 17,20 lyase activity (lane 4). Thus, although ROCK1 acts somewhere to increase 17,20 lyase activity and ROCK1 can phosphorylate P450c17 directly, this phosphorylation does not confer 17,20 lyase activity.

ROCK1 and P450c17 interact
Computational analysis for protein-protein interaction by protein interaction by structural matching (PRISM: http://gordion.hpc.eng.ku.edu.tr/prism/) predicts that P450c17 might interact with protein kinase N1 (PKN1) and ROCK1. Both protein kinase C-related kinase 1 (PRK1) and ROCK1 are members of the AGC group of kinases (cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family) (58) that can bind to, and function as, effectors for the small G protein, RhoA (59). However, unlike ROCK1, which can phosphorylate P450c17 in vitro (Fig. 3Go), active recombinant PKN1 (Invitrogen) did not phosphorylate P450c17 (data not shown). To determine whether ROCK1 interacts with P450c17, COS1 cells were cotransfected with P450c17 and ROCK1-wt and ROCK1-{Delta}3 constructs, immunoprecipitated with P450c17 antibody (24), and probed with ROCK1 antibody. As shown in Fig. 4AGo, both the full-length ROCK1 and the constitutively active form, ROCK1-{Delta}3, co-immunoprecipitated with P450c17 antibody. In NCI-H295A cells, we detected increased amounts of ROCK1 that co-immunoprecipitated with P450c17 under serum deprivation conditions (lane 1 vs. 2, Fig. 4BGo). ROCK1 probably interacted directly with P450c17. This interaction likely occurred at the catalytic domain of ROCK1, because in vitro pull-down assays showed that purified, bacterially expressed P450c17 could interact with a truncated form of recombinant ROCK1 containing residues 17–535 (Invitrogen), which contains only the catalytic domain (Fig. 4CGo).


Figure 4
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  		FIG. 4. Serum deprivation increases ROCK1-P450c17 interaction, P450c17 phosphorylation, and 17,20 lyase activity. A, COS-1 cells were transfected with P450c17 alone (lane 3) or with ROCK1-{Delta}3 (lane 2) and ROCK1-wt (lane 1). Cell lysates were immunoprecipitated (IP) with P450c17 antibody (lanes 1–3), and Western blot was done using ROCK1 antibody. B, NCI-H295A cells were incubated with and without serum for 4 h (lanes 1 and 3 vs. 2 and 4, respectively), immunoprecipitated with P450c17 antibody (lanes 1 and 2), and probed with ROCK1 antibody. C, GST-tagged catalytically active recombinant ROCK1 was incubated with a purified His-tagged P450c17 in 20 mM HEPES, 20 mM MgCl2, 200 µM ATP for 20 min at 30 C. Preformed complexes in lanes 1–2 and 3–4 were captured onto GST and Ni-NTA beads, respectively, washed, and eluted, and the gel was stained by Coomassie Blue R250. In lanes 5–6 and 7–8, ROCK1 and P450c17 were prebound to GST and Ni-NTA beads, respectively. The prebound input ROCK1-GST (lane 5) was incubated with P450c17 (lane 6), whereas prebound P450c17-Ni-NTA (lane 7) was incubated with ROCK1 (lane 8).

 
ROCK1 suppression by siRNA inhibits 17,20 lyase activity
Because ROCK1 phosphorylation of P450c17 did not confer lyase activity in vitro, it was not clear whether ROCK1 and/or ROCK2 participate in the pathway of P450c17 phosphorylation. To determine whether ROCK1 or ROCK2 mediates P450c17 phosphorylation, we used RNA interference to suppress the expression of these two forms of Rho kinase. Lentiviruses expressing ROCK1 and ROCK2 siRNAs were prepared from HEK-293T cells and used to infect subconfluent NCI-H295A cells for 2 d. As shown in Fig. 5AGo, ROCK1 and ROCK2 expression was suppressed in NCI-H295A cells infected with ROCK1 and ROCK2 siRNAs (lanes 3 and 8). ROCK2 siRNA had no significant effect on 17{alpha}-hydroxylase and 17,20 lyase activities; however, the ratio of 17,20 lyase to 17{alpha}-hydroxylase activity was inhibited approximately 1.5-fold by ROCK1 siRNA, suggesting that ROCK1 plays a role in the P450c17 phosphorylation pathway.


Figure 5
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  		FIG. 5. Suppression of ROCK1 by RNA interference inhibits 17,20 lyase activity. A, To confirm the silencing of ROCK1 and ROCK2 by RNA interference, 40 cycles of RT-PCR were done using cDNAs from lentivirus-infected NCI-H295A cells; β-glucuronidase (GUS) was used as an internal control. Subconfluent cells were infected for 2 d with lentiviruses expressing siRNAs for ROCK1 (lanes 3, 7, and 11) and ROCK2 (lanes 4, 8, and 12), or with lentiviruses prepared from the vector alone (lanes 1, 5, and 9) or a construct expressing a scrambled sequence (lanes 2, 6, and 10). B, NCI-H295A cells infected with lentiviral constructs of vector alone (lanes 1 and 5), a scrambled sequence (lanes 2 and 6), ROCK1 siRNA (lanes 3 and 7), and ROCK2 siRNA (lanes 4 and 8) were subjected to hydroxylase (lanes 1–4) and lyase (lane 5–8) assays with substrates [14C]progesterone or [3H]17-hydroxypregnenolone respectively. C, The above experiment was done in triplicate to determine the effects of ROCK1 and ROCK2 siRNAs on the ratio of 17,20 lyase to 17{alpha}-hydroxylase activities in NCI-H295A cells. *, Significant difference from controls; P = 0.00070 for ROCK1 siRNA.

 
Site-directed mutagenesis
The consensus phosphorylation sites recognized by ROCK1 are R/KXXS and R/KXS (60, 61). Six serine residues in human P450c17, S94, S213, S234, S256, S258, and S273, fit these consensus sequences. Modeling of the structure of human P450c17 (62) shows that residues S94, S256, S258, and S273 are surface exposed in the redox-partner interacting region and hence would appear to be reasonable candidates for the relevant serine phosphorylation site on human P450c17. Because ROCK1 can phosphorylate P450c17 in vitro, we sought to determine whether any of these sites is important for P450c17 phosphorylation and lyase activity. Therefore, we mutagenized a human P450c17 expression vector, changing each of these serine residues to alanine (to prevent phosphorylation) or to aspartic acid and glutamic acid to mimic the size and charge of phosphoserine. These constructs were transfected into nonsteroidogenic COS-1 cells, and the 17{alpha}-hydroxylase and 17,20 lyase activities of the mutant forms of P450c17 were assayed. All of the P450c17 mutants catalyzed 17{alpha}-hydroxylase and 17,20 lyase activities at levels comparable to wild-type P450c17, except for S94A, which had lower 17{alpha}-hydroxylase and 17,20 lyase activities, and the three mutants of S273, which produced approximately 1.5-fold higher 17,20 lyase activities (Table 5Go). However, in the presence of the ROCK1 inhibitor H-1152, the aspartic acid and glutamic acid mutants were not constitutively active because their lyase activities were inhibited approximately 30–60%, similar to that of the wild-type construct. Thus, S94, S256, S258, and S273 are not biologically important for P450c17 phosphorylation and 17,20 lyase activity.


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  		TABLE 5. Effects of mutations of candidate sites for phosphorylation of P450c17

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human P450c17 can be phosphorylated on serine and threonine (but not tyrosine) residues, and this phosphorylation augments the 17,20 lyase activity but not the 17{alpha}-hydroxylase activity (27, 29, 51). The search for the kinase(s) has been daunting. Human P450c17 contains 509 residues, of which 32 are serine and 25 are threonine (14). Computational analysis of the linear sequence of P450c17 with NetPhos (<http://www.cbs.dtu.dk/>www.cbs.dtu.dk), MotifScan (<http://scansite.mit.edu/cgi-bin/motifscan_seq>http://scansite.mit.edu/cgi-bin/motifscan_seq), and KinasePhos (<http://kinasephos.mbc.nctu.edu.tw/>http://kinasephos.mbc.nctu.edu.tw) indicate that 18 serines and 15 threonines are likely targets of kinases. Imposing these analyses on the computational model of the three-dimensional structure of P450c17 (62) indicates that most of these residues are predicted to be surface exposed and hence potentially accessible to kinases. These analyses suggest that the likely kinases include PKA, PKC, CaMII, casein kinase (CK)I/II, and PKB. However, these computationally based analyses are biased because they are based on databases of other proteins that are known to be phosphorylated; hence, the kinases and sites that have been most intensively studied are statistically overrepresented.

Kinase cascades related to the insulin and/or IGF-I signaling systems are attractive candidates because of many reported connections suggesting insulin induction of ovarian androgen synthesis and because a connection to the IGF-I system seems plausible (27). Studies in ovarian theca cells suggest a pathway that includes the PIK3/Akt pathway (42). The computational search indicated that S256 and S258, which lie on the edge of the redox-partner binding site, should be phosphorylated by the Akt-responsive kinase p70S6K. However, p70S6K phosphorylated P450c17 poorly in vitro, did not alter the 17,20 lyase activity, and was not found in NCI-H295A cells. Others have similarly postulated, examined, and excluded Ser258 and Thr260 as the relevant targets of phosphorylation (63). The ERK1/2 system appears to be activated in PCOS muscle cells (43), but ERK1/2 decreases androgen production in PCOS theca cells (64), and our microarray data show that neither ERK1 nor ERK2 were found in NCI-H295A cells; hence, this system does not appear to be involved in P450c17 phosphorylation.

ROCK1 is a serine/threonine kinase that phosphorylates the small G protein RhoA. The principal known role of ROCK1 is in regulating the force and velocity of actin/myosin cross-bridging (65), but ROCK1 also inhibits insulin signal transduction by catalyzing serine phosphorylation of IRS-1 (66) and hence is a plausible candidate for a factor associated with PCOS. COS-1 cells cotransfected with vectors overexpressing P450c17 and either full-length ROCK1 or C-terminally deleted (activated) ROCK1 increased the ratio of 17,20 lyase to 17{alpha}-hydroxylase activity 1.5-fold compared with control cells, but phosphorylation of bacterially expressed P450c17 did not affect its 17,20 lyase activity, even though addition of cytochrome b5 did increase 17,20 lyase activity, indicating that the P450c17 preparation was catalytically active. However, ROCK1 did catalyze incorporation of 32P into P450c17, GST pull-down experiments showed that ROCK1 and P450c17 interact in vitro, and use of a vector expressing myc-tagged ROCK1 shows that they interact in cells. These results suggest that ROCK1 may be involved in the phosphorylation of P450c17 but that its action is indirect. Thus, ROCK1 may act upstream in a pathway leading to the kinase that phosphorylates P450c17, or it may act as a priming kinase that permits a subsequent kinase to recognize P450c17 and phosphorylate it at a different site, which affects 17,20 lyase activity.


   Acknowledgments
 
We thank Dr. Christopher Barker of the UCSF Gladstone Institute Genomics Core Facility and Dr. Ru-Fang Yeh of the UCSF Center for Bioinformatics and Molecular Biostatistics for assistance with the microarray experiments, Drs. Christa Flück and Andrea Dardis for constructing some of the mutants of S256 and S258, and Ms. Isabella Damm and Mr. Michael Wongchaowart for technical assistance.


   Footnotes
 
This work was supported by National Institutes of Health Grants R01-HD41958 (to W.L.M.) and K08-DK64626 (to Q.D.) and by a UCSF School of Medicine Research Evaluation and Allocation Committee grant (to Q.D.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 10, 2008

Abbreviations: DHEA, Dehydroepiandrosterone; DHEAS, DHEA sulfate; GST, glutathione-S-transferase; 3β-HSD, 3β-hydroxysteroid dehydrogenase; IR, insulin receptor; IRS, insulin receptor substrate; MEK, MAPK/ERK kinase; mTOR, mammalian target of rapamycin; nt, nucleotides; P450c17, cytochrome P450c17; PCOS, polycystic ovary syndrome; PI3K, phosphatidylinositol-3-kinase; PKA, protein kinase A; PKN, protein kinase N; POR, P450 oxidoreductase; ROCK, Rho-associated, coiled-coil containing protein kinase; ROCK1-wt, wild-type ROCK1; siRNA, small interfering RNA; StAR, steroidogenic acute regulatory protein; TLC, thin-layer chromatography.

Received November 6, 2007.

Accepted for publication January 2, 2008.


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