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J. Biol. Chem., Vol. 278, Issue 5, 2837-2844, January 31, 2003

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Protein Phosphatase 2A and Phosphoprotein SET Regulate Androgen Production by P450c17^*

Amit V. Pandey, Synthia H. Mellon^§¶, and Walter L. Miller^¶

From the  Department of Pediatrics and ^§ Department of Obstetrics, Gynecology, and Reproductive Sciences, the ^¶ Metabolic Research Unit and the Center for Reproductive Sciences, University of California, San Francisco, California 94143-0978

Received for publication, September 17, 2002, and in revised form, November 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cytochrome P450c17 catalyzes 17-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Steroid hormones are synthesized by a set of pathways that begin with the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc, the quantitative regulator of steroidogenesis. Pregnenolone can then be directed to one of three principal pathways by microsomal P450c17, the qualitative regulator of steroidogenesis, which catalyzes both 17-hydroxylase and 17,20 lyase activities (1-4) (Fig. 1). In the absence of P450c17, e.g. in adrenal zona glomerulosa and ovarian granulosa cells, pregnenolone is converted to 17-desoxy, C21 steroids including progesterone, corticosterone, and aldosterone. In the presence of the 17-hydroxylase activity of P450c17, the adrenal zona fasciculata produces C21 17-hydroxy steroids including the glucocorticoid cortisol. When both 17-hydroxylase and 17,20 lyase activities are present, the adrenal zona reticularis and gonads produce the C19 17-ketosteroid dehydroepiandrosterone (DHEA),¹ which is the precursor of androgens and estrogens.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Early steps of sex steroid biosynthesis. P450scc converts cholesterol to pregnenolone, a C21 5-steroid. Human P450c17 performs the 17-hydroxylase reaction equally well using pregnenolone and progesterone as substrates, but the 17,20 lyase reaction occurs 50-100 times more efficiently using 17OH-Preg as substrate rather than 17OH-Prog. Thus conversion of 17OH-Prog to androstenedione is minimal, and DHEA is the principal precursor of sex steroid synthesis.

The ratio of the 17-hydroxylase to 17,20 lyase activities of human P450c17 is developmentally regulated. Adjusted for body size, the human adrenal produces nearly constant amounts of cortisol throughout life, indicating relatively constant 17-hydroxylase activity (5). By contrast, the production of DHEA is minimal in childhood, rises 100-fold to levels that exceed the production of cortisol in young adulthood, and then gradually decreases with advancing age (6). The mechanisms by which human adrenal C19 steroid synthesis is turned on (adrenarche) and turned off (adrenopause) remain unclear. Recent clinical observations suggest a link between premature exaggerated adrenarche and the polycystic ovary syndrome (7-10). Adrenarche is difficult to study because it occurs only in higher primates (10-12), and no cellular model exists; instead, a productive approach has centered on the biochemistry of P450c17.

P450c17 is a single enzyme encoded by a single gene that is expressed in both the adrenals and gonads (13, 14). Like other microsomal P450 enzymes, P450c17 catalyzes several chemical reactions on a single active site by receiving electrons from the flavoprotein P450 oxidoreductase. Human P450c17 can catalyze the 17-hydroxylation of pregnenolone to 17OH-pregnonolone (17OH-Preg) or of progesterone to 17OH progesterone (17OH-Prog). However, the 17,20 lyase reaction almost exclusively converts 17OH-Preg to DHEA; human P450c17 catalyzes the conversion of 17OH-Prog to androstenedione with only 3% of the efficiency of the reaction with 17OH-Preg (15); thus most human sex steroids are made from DHEA. At least three factors influence the ratio of 17,20 lyase to 17-hydroxylase activities at a post-translational level. First, high molar ratios of P450 oxidoreductase to P450c17 favor lyase activity (2, 15, 16). Second, cytochrome b₅ acts allosterically to foster the interaction between P450c17 and P450 oxidoreductase to promote the lyase reaction, but b₅ does not function as an electron donor (2, 15, 17). Third, the phosphorylation of P450c17 on serine and threonine but not tyrosine residues increases 17,20 lyase activity through as-yet-unidentified mechanisms, which may involve increasing its affinity for b₅ and/or P450 oxidoreductase (18). The precise residues of P450c17 that are phosphorylated and the responsible kinase(s) remain unknown. However, when a protein is activated by phosphorylation there generally is an equilibrium between phosphorylation by a kinase and dephosphorylation by a phosphatase (19). We now provide evidence that PP2A serves as the principal phosphatase regulating P450c17 phosphorylation and 17,20 lyase activity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cells and Transfection-- The NCI-H295A adherent population (20) of human adrenocortical NCI-H295 tumor cells (21, 22) was grown on 150-mm Petri plates as described (20). Cells at 60-80% confluence were treated with okadaic acid, fostriecin, and cantharidin (Calbiochem, www.calbiochem.com). NCI-H295A cells in 100-mm dishes were transfected with 5 µg of the plasmids pBJF, pBJF-Flag-PP2A, pBJF-Flag-PP4, or pBJF-Flag-PP6 (23) using LipofectAMINE 2000 (Invitrogen, www.invitrogen.com) following the manufacturer's protocol. For in vitro labeling with ³²P, NCI-H295A cells were transferred to phosphate-free medium for 1 h and labeled with 1 mCi/ml [³²P]orthophosphate for 4 h at 37 °C.

Microsome Preparation and Enzyme Assays-- NCI-H295A cells were harvested and lysed by sonication (six times for 10 s at 30 kC s¹) in 50 mM potassium phosphate buffer (pH 7.4), containing 100 mM KCl and 0.1 mM EDTA. Unbroken cells, the nuclear fraction, and mitochondria were separated at 12,000 × g for 20 min, and microsomes were pelleted from the 12,000 × g supernatant at 100,000 × g for 90 min. Microsomes were resuspended in the same buffer containing 20% glycerol and used immediately for enzyme assay. 17-Hydroxylase and 17,20 lyase activity assays were as described (15, 24). Briefly, microsomes (20 µg of protein) were incubated at 37 °C with 50,000 cpm of [³H]progesterone or [³H]17OH-Preg in 50 mM potassium phosphate buffer (pH 7.4), and catalysis was initiated by addition of 1 mM NADPH. After the appropriate time (30-60 min), steroids were extracted in 500 µl of 1:1 ethyl acetate/isooctane and concentrated by evaporation under nitrogen. Concentrated steroids were dissolved in 20 µl of trichloromethane and analyzed by thin layer chromatography over silica gel plates (PE SIL G/UV, Whatman) using a 3:1 mixture of chloroform/ethyl acetate as a mobile phase. Radiolabeled steroids were quantitated using a Storm 860 PhosphorImager (Amersham Biosciences, www.amershambiosciences.com).

Coimmunoprecipitation and Western Blotting-- Antibodies against PP2A catalytic subunit (Upstate Biotechnology, Inc., www.upstatebiotech.com), PP4 (25), and SET (26) were covalently linked to protein A-Sepharose using disuccinimidyl suberate (Pierce, www.piercenet.com) and mixed with lysates of NCI-H295A cells (4 µg of antibody/ml of lysate). The bound proteins were eluted, separated by SDS, 4-20% PAGE, immunoblotted with P450c17 antiserum (16) (1:3000 dilution), and detected by enhanced chemiluminescence (Amersham Biosciences).

Phosphatase Assays-- NCI-H295A cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 120 mM NaCl, 1 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin and leupeptin. Recombinant catalytic subunit of PP2A was from Promega (www.promega.com); PP4 was prepared by immunoprecipitation from NCI-H295A lysates using antibody provided by Dr. T. H. Tan (25). The activities of these phosphatases were confirmed against phosphorylated microsomal proteins by monitoring the release of free phosphate. PP4 and PP2A were immunoprecipitated and washed three times with 50 mM HEPES buffer (pH 7.4) containing 0.1% Triton X-100 and 500 mM NaCl. Immunoprecipitates were incubated in 100 µl of 50 mM HEPES (pH 7.0), 0.1 mM DTT, 0.1 mM CaCl₂, 1 mM MgCl₂, and 1 mM MnCl₂ at 25 °C for 30 min and pelleted by centrifugation, and the supernatant was transferred to fresh tubes. The assay was terminated with 500 µl of Biomol Green reagent (Biomol, www.biomol.com), a phosphate detection reagent based on malachite green, incubated for 20 min at room temperature, and read at 620 nm. One unit of phosphatase activity was defined as the amount of enzyme required to release 1 nmol of phosphate per h at 30 °C. For the in vitro PP2A experiments, microsomes were treated with 12.5 units/ml of pure recombinant PP2A catalytic subunit (Promega) at 25 °C in 50 mM Tris-HCl (pH 7.4) containing 20 mM MgCl₂. The reaction was terminated by addition of 1 µM okadaic acid and 10 mM NaF and chilling on ice for 10 min.

siRNA Construction and Transfection-- Small interfering RNAs (siRNAs) to the catalytic subunits of human PP2A and PP4 were designed with 3'-overhanging thymidine dimers as described (27). Target sequences were aligned to the human genome data base in a BLAST search (www.ncbi.nlm.nih.gov/blast) to eliminate those with significant similarity to other genes. Web-based siRNA design software from Ambion (www.ambion.com/techlib/misc/siRNA_finder.html) was used for selecting siRNA sequences. Three target sequences for each gene corresponding to sequences located in the 5', 3', or middle regions of each transcript were synthesized and used for transfection (Table I). The siRNAs were synthesized using a transcription-based Silencer^TM siRNA synthesis kit (Ambion, www.ambion.com). Transfection was carried out using the OligofectAMINE transfection reagent (Invitrogen) as described (28). In brief, NCI-H295A cells were grown to 50-70% confluency in complete medium without antibiotics in 100-mm plates. Cells were washed with serum-free medium prior to transfection. All the siRNA duplexes (2.0 µg/100-mm plate) were diluted in separate tubes with 100 µl of Opti-MEM^TM (Invitrogen). In a separate tube, OligofectAMINE reagent was diluted 1:5 with Opti-MEM^TM medium and incubated for 10 min at room temperature. Diluted OligofectAMINE reagent was added to siRNA duplexes (50 µl of diluted OligofectAMINE reagent/µg of siRNA), and the mixture was incubated for 20 min at room temperature. The volume of medium overlaying the cells was adjusted to 7.5 ml, and the siRNA transfection complexes (200 µl) were added; 6 h later the medium was supplemented with serum to make a final serum concentration of 2%. Because of the slow doubling time of NCI-H295A cells (20, 22), the cells were then incubated for an additional 72 h before harvesting for analysis.

Preparation and Transfection of Rat SET Protein-- Full-length rat SET cDNA (29) was subcloned into pBluebac His2Sf9 (Invitrogen) and transfected into Spodoptera Sf9 cells for 48 h. Cells (50 ml of culture) were collected, washed, and lysed in 4 ml of 500 mM NaCl, 25 mM HEPES (pH 7.5) (buffer B) containing 1% Tween 20, 10% glycerol, 1 mM pefabloc, and a mixture of protease inhibitors. The cell lysate was applied to a 250-µl Ni-NTA column previously equilibrated with buffer B containing 1% Tween 20. The column was first washed with 5 ml of buffer B containing 1% Tween 20, then with 5 ml of buffer B, and finally with 5 ml of buffer B containing 5 mM imidazole. The bound SET was eluted with 5 ml of buffer B containing 250 mM imidazole. Protein transfection into NCI-H295A cells was performed with Chariot reagent (Active Motif, www.activemotif.com). NCI-H295A cells were plated in 6-well plates and grown to 40-60% confluency. Purified SET protein was diluted with PBS (1.0 µg/ml), and Chariot reagent was diluted with water (1:20). In a separate tube, 100 µl of diluted SET protein was mixed with 100 µl of diluted Chariot reagent and incubated at room temperature for 30 min. Growth medium from the cells was aspirated, and cells were washed with PBS. Transfection complex (200 µl/well) was added to cells; the volume was adjusted to 600 µl with serum-free RPMI 1640 medium, and the cells were incubated for 1 h (37 °C, 5% CO₂). Cells were supplemented with 1.0 ml of complete growth medium, and incubation was continued for an additional 4 h after which the cells were harvested and used for experiments.

PCR of Human SET and Phosphatases-- Total RNA (1.0 µg) from NCI-H295A cells was converted to cDNA using Superscript II reverse transcriptase (Invitrogen) for 30 min at 50 °C, followed by 35 cycles of PCR amplification (30 s at 94 °C, 30 s at 60 °C, and 90 s at 72 °C). Primer pairs are described in Table II.

Preparation of Endogenous Kinases and in Vitro Phosphorylation-- Soluble intracellular kinases were enriched using an ATP-Sepharose matrix (Upstate Biotechnology, Inc.) as described (30). ATP-Sepharose matrix (0.25 ml) was washed three times with 1 ml of 25 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM NADH, 1 mM NAD, 1 mM ADP, 1 mM AMP, 1 mM DTT, and 60 mM MgCl₂ (buffer A). NCI-H295A cells were lysed in 50 mM Tris-HCl (pH 7.4) containing 1% Nonidet P-40; 5 mM EDTA; 2 mM EGTA; 150 mM NaCl; 1 mM phenylmethylsulfonyl fluoride; 1 µg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM DTT; 0.15 mM Na₃VO₄; 10 mM NaF; 500 µM cantharidin; 1 mM NADH; 1 mM NAD; 1 mM ADP; and 1 mM AMP and centrifuged at 100,000 × g for 90 min at 4 °C; and 1 ml of the soluble fraction (1 mg of protein) was added to the washed ATP-Sepharose beads. After incubation for 4 h with agitation at 4 °C, the beads were washed 4 times with 0.5 ml of buffer A containing 500 mM NaCl, suspended in 0.5 ml of buffer A containing 10 mM ATP, and incubated at room temperature for 60 min to elute bound kinases. Bacterially expressed human P450c17 bound to Ni-NTA-Sepharose was incubated with 1-2 µg of protein from the kinase-enriched fraction of NCI-H295A cells in the presence of 10 mM Mg-ATP. NTA-Sepharose containing 0.25-0.5 µg of bound P450c17 in a volume of 50 µl was incubated with 1 mM [³²P]ATP (1.0 µCi) and 25 mM MgCl₂ and washed 5 times with 0.5 ml of 50 mM Tris-HCl (pH 7.4) containing 500 mM NaCl. Bound ³²P-P450c17 was denatured by boiling in SDS gel sample buffer, separated by SDS-12% PAGE, and analyzed by PhosphorImager.

Bacterially Expressed Human P450c17-- The pCWH17-mod(His)4 expression plasmid containing the cDNA for modified human P450c17 (31) was transformed into Escherichia coli JM109. Ampicillin-resistant colonies were grown at 37 °C to A₆₀₀ 0.4-0.6; P450c17 expression was induced by 0.4 mM isopropyl-1-thio--D-galactopyranoside at 25 °C for 48 h, and P450c17 was purified as described (31). In brief, spheroplasts prepared by lysozyme treatment of bacteria were lysed by sonication for 3 min at 30 kC s¹ and centrifuged at 4,000 × g for 10 min, and the pellet containing P450c17 was extracted with 1% Triton X-114 (Calbiochem) and ultracentrifuged at 100,000 × g for 30 min. A reddish brown detergent-rich supernatant fraction containing P450c17 was isolated and passed over a Ni-NTA-Sepharose column. The column was washed with 20 mM histidine to remove nonspecific binding and eluted with 200 mM histidine. Further purification was carried out by hydroxyapatite chromatography to remove histidine and some other protein contaminants.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Regulation of 17,20 Lyase Activity by Phosphatases in NCI-H295A Cells-- Serine/threonine phosphorylation of P450c17 fosters 17,20 lyase activity, and dephosphorylation of P450c17 in vitro with alkaline phosphatase decreases 17,20 lyase activity (18). To determine whether a phosphatase participates in the physiological regulation of 17,20 lyase activity in vivo, we treated NCI-H295A cells with phosphatase inhibitors and measured 17,20 lyase activity (Table III). Okadaic acid (32, 33), cantharidin (34), and fostriecin (35) are inhibitors of protein phosphatases 2A (PP2A) and 4 (PP4) (36). Low concentrations of okadaic acid and cantharidin that are relatively specific for PP2A and PP4 increased 17,20 lyase activity 4-fold. Okadaic acid and cantharidin have some activity against protein phosphatase 1 (PP1) (35), but fostriecin has IC₅₀ values of 1.5 nM for PP2A (35, 36), 3.0 nM for PP4 (36), and 131 µM for PP1 (35), making it an essentially pure inhibitor of PP2A and PP4 (35, 36). Concentrations of either 5 or 25 nM fostriecin again increased 17,20 lyase activity 4-fold, implicating PP2A and/or PP4 as the relevant phosphatases.

The action of okadaic acid on NCI-H295A cells was dose-dependent with a half-maximal effect on lyase activity at 10 nM and a maximal effect at 100 nM, but there was no effect on 17-hydroxylase activity (Fig. 2A). Although it is logical to presume that the action of PP2A to inhibit 17,20 lyase activity was directly attributable to dephosphorylation of P450c17, it could also have resulted from an indirect protein-protein interaction. To discriminate between these two possibilities, PP2A was preincubated with various concentrations of okadaic acid. The okadaic acid-treated PP2A was added to NCI-H295A microsomes, and the 17,20 lyase activity was measured (Fig. 2B). The half-maximal inhibitory concentration of okadaic acid was ~0.5 nM, suggesting that catalytic activity of PP2A is required for its inhibition of 17,20 lyase activity. Preincubation of PP2A with 100 nM okadaic acid for 15 min before addition to microsomal preparations neutralized the effect of PP2A (data not shown), consistent with PP2A inhibiting 17,20 lyase activity by dephosphorylating P450c17. These values are consistent with the IC₅₀ value of okadaic acid (0.1 nM) on purified preparations of PP2A or PP4 and those observed in cell culture treatments (10 nM) (32). Thus PP2A or PP4 appear to function physiologically as intracellular factors that de-phosphorylate P450c17, resulting in suppression of 17,20 lyase activity and sex steroid synthesis.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Okadaic acid and phosphatase treatment of NCI-H295A cell microsomes. A, okadaic acid increases 17,20 lyase activity in NCI-H295A cells. After incubating cells for 60 min with the indicated concentrations of okadaic acid, cells were harvested, and microsomes were prepared. 17-Hydroxylase activity was measured as the conversion of [3H]progesterone to 17OH-Prog (open bars) and 17,20 lyase activity was measured as the conversion of [3H]17OH-Preg to DHEA (closed bars), assayed by thin layer chromatography. The data are shown normalized to controls with no okadaic acid. The specific activity for the control 17-hydroxylase reaction was 0.25 pmol of 17OH-Preg produced per µg of protein per min, and for 17,20 lyase reaction it was 0.35 pmol of DHEA produced per µg of protein per min. B, PP2A treatment of microsomes in the presence of okadaic acid. Microsomes isolated from NCI-H295A cells were treated with 5.0 units/ml of PP2A in the presence of the indicated concentrations of okadaic acid. Data are presented as % of activity seen in the absence of okadaic acid; black bars, without PP2A treatment; white bars, with PP2A treatment. C, effect of okadaic acid (OA) on phosphorylation of P450c17. NCI-H295A cells grown in 32P were treated with okadaic acid, and the labeled P450c17 was immunoprecipitated with antiserum bound to protein A-Sepharose beads and analyzed on SDS-12% PAGE by Western blotting (top) and PhosphorImaging (bottom).

To determine whether the induction of 17,20 lyase activity by phosphatase inhibitors correlated with the degree of P450c17 phosphorylation, we grew NCI-H295A cells in ³²P and okadaic acid and estimated ³²P incorporation into P450c17. PhosphorImaging of equivalent amounts of immunoprecipitable P450c17 showed that 10 nM okadaic acid promoted the incorporation of ³²P (Fig. 2C). Therefore, phosphatases affected by okadaic acid (PP2A/PP4) appear to play a role in the reversible phosphorylation of P450c17.

Effect of PP2A and PP4 on P450c17 Activities in NCI-H295A Microsomes-- To identify the specific phosphatases responsible for the effects of the inhibitor treatments, we treated steroidogenically active microsomes from NCI-H295A cells with PP2A and PP4 and assayed 17,20 lyase activity. PP2A (12.5 units/ml) inhibited 17,20 lyase activity to the same extent as alkaline phosphatase (12.5 units/ml), but PP4 (25 units/ml) had no effect (Fig. 3A). This action of PP2A could be partially overcome by pretreating the PP2A with the phosphoprotein SET (7.8 nM), an inhibitor of PP2A (37). SET alone did not affect 17,20 lyase activity (not shown). To determine whether PP2A was sufficient for removing the physiologically relevant phosphate groups, we treated NCI-H295A microsomes with PP2A (Fig. 3B). Preincubation of microsomes with up to 12.5 units/ml of PP2A had no effect on the conversion of progesterone to 17OH-Prog (an index of 17-hydroxylase activity), but PP2A decreased 17,20 lyase activity (conversion of 17OH-Preg to DHEA) in a dose-dependent manner with a half-maximal effect at about 0.5 units/ml and complete inhibition at 12.5 units/ml. Thus PP2A exerts the same selective effect on the steroid 17,20 lyase activity of P450c17 that we previously observed with nonspecific calf intestinal alkaline phosphatase (18), but PP4 does not exert this effect. The presence of PP2A, PP4, and SET in NCI-H295A cells was confirmed by Western blotting (data not shown).

View larger version (59K): [in this window] [in a new window]   Fig. 3.   Effect of protein phosphatases on P450c17 activities. A, effect of phosphatases on 17,20 lyase activity. NCI-H295A microsomes were treated as indicated, and conversion of 17-OH Preg to DHEA was assessed by thin layer chromatography. PP4 did not inhibit 17,20 lyase activity, whereas both alkaline phosphatase and PP2A did. The effect of PP2A could be partially overcome by preincubating PP2A with SET. PP2A and PP4 used could dephosphorylate other microsomal proteins in vitro (not shown). The activity of SET was confirmed by its ability to inhibit PP2A. The numbers in parentheses indicate activity as a percentage of control from quantitation by PhosphorImager. The specific activity for the 17,20 lyase reaction in control cells was 0.35 pmol of DHEA produced per µg of protein per min. B, response of P450c17 enzyme activities to treatment with PP2A. Treatment of microsomes from NCI-H295A cells with the indicated amounts of PP2A had no detectable effect on 17-hydroxylase activity (conversion of progesterone to 17OH-Prog), but PP2A inhibited 17,20 lyase activity in a dose-dependent manner (conversion of 17OH-Preg to DHEA) with an IC50 value of 2.0 units/ml (mean of three experiments). The numbers in parentheses indicate activity as a percentage of control from the quantitation of data by PhosphorImager. The specific activity for the 17,20 lyase reaction was 0.35 pmol of DHEA produced per µg of protein per min, and for the 17-hydroxylase reaction, 0.25 pmol of 17OH-Preg produced per µg of protein per min.

PP4 and PP6 Cannot Mimic the Action of PP2A-- PP2A shares 66% sequence identity with PP4 and 58% identity with PP6, indicating they belong to a related family of phosphatases (38), and some inhibitors of PP2A also affect PP4 (36, 38) and might potentially inhibit PP6. To determine whether the action of PP2A on P450c17 was specific or simply representative of this family of phosphatases, we transfected NCI-H295A cells with expression vectors for the catalytic subunits of PP2A, PP4, or PP6 (23), verified the expression of these proteins by Western blotting, and measured 17,20 lyase activity (Fig. 4A). Compared with cells transfected with an empty vector, the 17,20 lyase activity of cells expressing PP2A was reduced, whereas the 17,20 lyase activity of cells expressing PP4 and PP6 was unchanged. Thus PP4 and PP6 were unable to dephosphorylate the relevant residues of P450c17.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Effect of phosphatases on P450c17. A, effect of PP2A, PP4, and PP6 on 17,20 lyase activity. NCI-H295A cells were transfected for 36 h with expression vectors for the catalytic subunits of PP2A, PP4, PP6, or empty vector control, and 17,20 lyase activity was measured in isolated microsomes. PP2A but not PP4 and PP6 reduced 17,20 lyase activity. The numbers in parentheses indicate activity as a percentage of control from the quantitation of data by PhosphorImager. B, coimmunoprecipitation of P450c17 with phosphatases. NCI-H295A cell lysates were immunoprecipitated with antisera to PP2A, PP4, and SET, separated by SDS-12% PAGE, and probed by Western blotting with antiserum to P450c17. NCI-H295A cell lysate contains immunodetectable P450c17, confirmed by the comigration with purified recombinant P450c17. Cell lysates immunoprecipitated with antiserum to PP4 or SET did not coimmunoprecipitate P450c17, but lysates immunoprecipitated with antisera to PP2A did. Western blot analysis of immunoprecipitates show that antisera against PP2A, PP4, and SET bring down these proteins.

To determine whether PP2A interacts directly with P450c17, we immunoprecipitated PP2A, PP4, and SET from NCI-H295A cells under non-denaturing conditions, and we confirmed the immunoprecipitation of each by Western blotting (not shown). Probing with antisera to P450c17 showed that P450c17 coimmunoprecipitated with PP2A but not with PP4 or SET (Fig. 4B), indicating that the action of PP2A is to dephosphorylate P450c17 itself and not some other protein that influences P450c17 activity.

Suppression of PP2A by siRNA-- To determine whether the effects of PP2A on the 17,20 lyase activity that we had documented with the biochemical assays in vitro were relevant to the regulation of 17,20 lyase activity in cells in vivo, we used RNA interference to suppress the expression of the catalytic subunits of PP2A and PP4 in NCI-H295A cells. Three 21-nucleotide siRNA segments directed against the 5' end, the middle, or the 3' end of the mRNAs for PP2A and PP4 were transfected into NCI-H295A cells, and the cells were examined 72 h later. Western blotting showed that both PP2A and PP4 were reduced by half compared with cells transfected with control 21-nucleotide RNAs (Fig. 5A). Neither transfection with the control RNA nor the siRNA directed against PP4 affected 17,20 lyase activity, but transfection with siRNA against PP2A increased 17,20 lyase activity by 250% (Fig. 5, B and C). Thus PP2A regulates 17,20 lyase activity in vivo as well as in vitro.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of PP2A and PP4 suppression by siRNA on 17,20 lyase activity. A, upper panels, western blots of PP2A and PP4 in NCI-H295A cells 72 h after transfection with siRNAs. Lower panels, thin layer chromatograms showing increased 17,20 lyase activity of NCI-H295A cells transfected with siRNA against PP2A, but not with siRNA directed against PP4, as compared with untransfected cells and cells transfected with a control (Cont) siRNA of the same length. B, suppression of PP2A but not PP4 leads to increased 17,20 lyase activity in NCI-H295A cells. NCI-H295A cells were transfected with siRNAs against PP2A or PP4, and 17,20 lyase activity was measured in isolated microsomes. Suppression of PP2A but not PP4 resulted in increased 17,20 lyase activity.

Role of SET in Regulating 17,20 Lyase Activity-- The presence of both a kinase and PP2A in NCI-H295A cells indicates the presence of conflicting activities, suggesting that each activity may be regulated by a cascade of additional factors. The phosphoprotein SET, a highly specific inhibitor of PP2A (37), was tested as an attractive candidate for such a factor. SET exerts other activities, including inhibition of cell cycle (39, 40), promoting expression of the gene for P450c17 in mouse testis MA-10 Leydig cells (29, 41) and modifying the substrate specificity of PP1 (42). SET (7.8 nM) inhibited the activity of PP2A but not PP4 using phosphorylated NCI-H295A microsomal proteins as substrate in vitro (Fig. 6A). Because transfection of SET expression vectors inhibited cell cycle, we assayed the activity of SET on P450c17 in vivo using a liposome-mediated protein transfection procedure to introduce recombinant rat SET into NCI-H295A cells (65-75% transfection efficiency; data not shown). Transfection with 25 ng of SET for 4 h increased 17,20 lyase activity to the same degree as treating the cells with 10 nM okadaic acid (Fig. 6B) but had no effect on the total amount of P450c17 protein detectable by Western blot (Fig. 6C). Analysis of NCI-H295A cells by RT-PCR shows that they express a wide variety of protein phosphatases (Fig. 6D), and they express both SET and SET (Fig. 6E), which differ only at their amino termini, due to alternate first exon choice (43); both SET and SET can inhibit PP2A activity equally in vitro (44).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of SET protein on P450c17. A, SET inhibits PP2A but not PP4. Purified preparations of PP2A and PP4 were preincubated with or without SET for 15 min; phosphorylated NCI-H295A microsomal proteins were added, and the amount of free phosphate released into solution was quantitated at 620 nm using an assay system based on malachite green. SET inhibited the activity of PP2A but not PP4. Data are mean ± S.D. of triplicate assays. B, SET fosters 17,20 lyase activity. Equal masses of protein (SET or bovine serum albumin) were introduced into NCI-H295A cells by protein transfection and then microsomes were isolated and assayed for 17,20 lyase activity. Data are mean ± S.D. from three different experiments. Specific activity for the 17,20 lyase reaction in control cells was 0.35 pmol of DHEA produced per µg of protein per min. C, Western blot showing that the amount of P450c17 did not change in cells transfected with SET as compared with controls or cells treated with okadaic acid (OA). D, RT-PCR of phosphatases in NCI-H295A cells. E, RT-PCR of SET isoforms in NCI-H295A cells. RT-PCR was performed using 1 µg of total RNA from NCI-H295A cells for cDNA synthesis using gene-specific primers, followed by 35 cycles of amplification and displayed by agarose gel electrophoresis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Reactivation of 17,20 Lyase Activity in NCI-H295A Microsomes Treated with PP2A-- To confirm that the action of PP2A on 17,20 lyase activity is mediated by dephosphorylating P450c17 rather than by working on some other upstream target, we determined if the 17,20 lyase activity that had been lost to the action of PP2A could be restored by re-phosphorylating P450c17. We prepared NCI-H295A cytoplasmic extract and enriched it for protein kinase activity by affinity chromatography on ATP-Sepharose (30). The retained fraction lacked phosphatase activity but was enriched for ATP-dependent kinase activity. NCI-H295A microsomes were dephosphorylated with PP2A and then the PP2A was inactivated with 100 nM okadaic acid and 10 mM NaF. Under these conditions, the microsomes retained 17-hydroxylase activity but had lost almost all 17,20 lyase activity (Fig. 7A). Re-phosphorylation of these PP2A-treated microsomes using 10 mM Mg-ATP and the cytosolic fraction enriched for protein kinases (5-10 µg of protein) fully restored 17,20 lyase activity (Fig. 7A). Neither the kinase preparation nor the cytosolic fraction of NCI-H295A cells contained significant 17,20 lyase activity (Fig. 7B). Thus one or more ATP-dependent protein kinases present endogenously in NCI-H295A cell cytoplasm is sufficient to restore full 17,20 lyase activity to dephosphorylated P450c17 in NCI-H295A microsomes.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of endogenous kinase(s) and PP2A on P450c17. A, endogenous kinases in NCI-H295A cells can foster 17,20 lyase activity. NCI-H295A microsomes were assayed for 17,20 lyase activity after treatment with the preparations indicated; activity is shown as the percent of activity seen with untreated microsomes (left-hand bar). The specific activity for the 17,20 lyase reaction in control cells was 0.35 pmol of DHEA produced per µg of protein per min. Kinase refers to the kinase-enriched fraction eluted from the ATP-Sepharose matrix. Endogenous kinases were able to reverse the effect of PP2A on 17,20 lyase activity, confirming the reversible nature protein phosphorylation in P450c17. B, the lyase activity of NCI-H295A cells is confined to microsomes (Mic) and is not found in cytosol or the kinase fraction. C, in vitro phosphorylation of bacterially expressed human P450c17. Bacterially expressed modified human P450c17 bound to Ni-NTA-Sepharose was incubated with the kinase fraction from NCI-H295A cells plus [32P]ATP (lane 2). Controls of P450c17 plus ATP without the kinase fraction or of the kinase fraction plus ATP without P450c17 (not shown) showed low incorporation or binding of 32P. Incubation of P450c17 with the kinase fraction showed substantial incorporation of 32P (lane 2), a significant part of which could be removed with PP2A (lane 4). Lane 1 shows molecular weight markers. D, in vitro phosphorylation of P450c17 immunoprecipitated from NCI-H295A cells. Immunoprecipitated P450c17 was incubated with the kinase (Kin) fraction from NCI-H295A cells plus [32P]ATP. Incorporation of 32P into P450c17 was increased in presence of the kinase fraction from NCI-H295A cells.

To determine whether NCI-H295A microsomes contain other unidentified factors required for the phosphorylation of P450c17, we examined the ability of the NCI-H295A cytosolic kinases to phosphorylate purified human P450c17 expressed in bacteria (31). Human P450c17 containing amino-terminal modifications that confer solubility without affecting activity (31) was expressed in E. coli JM109 cells and purified by Ni-NTA-Sepharose chromatography. P450c17 (0.25-0.5 µg), still attached to Ni-NTA-Sepharose through a His₄ linker, was incubated at 25 °C for 30 min with 1 µCi of [³²P]ATP (1 mM), 25 mM MgCl₂, and the cytoplasmic kinase fraction was prepared from NCI-H295A cells (1-2 µg of protein). The Ni-NTA-Sepharose-P450c17 beads were washed to remove other proteins and separated by 12% SDS-PAGE, and ³²P incorporation in P450c17 bands was detected by PhosphorImager analysis. The endogenous kinases present in NCI-H295A cytoplasm could phosphorylate P450c17 (Fig. 7C). Comparison of the amount of ³²P incorporated with the amount of P450c17 protein indicated that an average of 5.7 phosphates was incorporated. Treatment with PP2A diminished the acquired radioactivity confirming that PP2A acts to dephosphorylate P450c17. Equivalent results were obtained using P450c17 immunoprecipitated from NCI-H295A microsomes (Fig. 7D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reversible protein phosphorylation by protein kinases and phosphatases regulates numerous cellular processes. About 30% of proteins undergo phosphorylation that influences their conformation and biological activity (38, 45). Although most studies of protein phosphorylation have focused on protein kinases, recent data suggest that phosphatases are equally important components of reversible regulatory cycles of phosphorylation and dephosphorylation (19, 45). Based on primary amino acid sequences and three-dimensional structures, there are three main families of protein phosphatases, termed PPP, PPM, and PTP (38). The PPP and PPM families are serine/threonine phosphatases, whereas the PTP phosphatases can dephosphorylate tyrosine as well as serine and threonine. PP1, PP2A (also called PP2), PP2B (also called PP3), PP4, PP5, PP6, and PP7 are the principal members of the PPP family. PP2A is a heterotrimer of A, B, and C subunits (46). The 36-kDa catalytic C subunit attaches to one of over 50 different B subunits ranging from 50 to 130 kDa, with the help of the 65-kDa A subunit (19). Different PP2A holoenzymes form at various phases of the cell cycle and during metabolic processes (19, 46, 47). Okadaic acid, microcystin LR, tautomycin, cantharidin, calyculin A, and fostriecin are highly specific inhibitors of the PPP family (36, 38, 48). The sensitivity of specific cellular process to different concentrations of these inhibitors can facilitate the identification of physiologically relevant phosphatases. Okadaic acid and fostriecin are particularly useful as they can permeate membranes, inhibiting PP1, PP2A, and PP4 in intact cells (33, 34, 38). The sensitivity of the 17,20 lyase reaction to very low concentrations of fostriecin and okadaic acid, both in vivo and in vitro, the sensitivity to SET protein, the inability of PP4 and PP6 to dephosphorylate and inhibit the 17,20 lyase reaction, and the specific effect of suppression of PP2A, but not PP4, by siRNA all strongly suggest a role for PP2A in the regulation of P450c17 phosphorylation and 17,20 lyase activity.

PP2A activity can be regulated by a tyrosine kinase that phosphorylates and inactivates the catalytic subunit (49) and by interaction with SET (37). SET is also a nuclear protein involved in cell proliferation and inhibiting cyclins (39) and is also known as TAF-1, which regulates adenovirus DNA replication (50). SET also acts as a transcription factor that regulates mouse testicular P450c17 (29, 41). SET appears to be an endogenous regulator of the action of PP2A on P450c17. SET has been identified as the heat-stable cytoplasmic peptide inhibitor of PP2A termed I₂^PP2A (37). NCI-H295A cells express SET endogenously; transfection of these cells with recombinant SET protein enhanced 17,20 lyase activity similarly to the effect of okadaic acid, and SET did not inhibit PP4, implicating the PP2A/SET system as regulating the 17,20 lyase activity of P450c17. SET itself is a phosphoprotein. It is possible that phosphorylation/dephosphorylation of SET by other protein kinases and phosphatases governs its function as an inhibitor of PP2A, providing another site for control of the 17,20 lyase activity of P450c17. It is not yet clear if SET is involved in the transcriptional regulation of the human gene for P450c17 as it is with mouse gene. We found no change in the amount of P450c17 protein in our experiments, consistent with post-translational action. The regulation of 17,20 lyase activity at both the transcriptional and post-translational level by a single protein would make it an attractive candidate for a factor involved in adrenarche and the hyperandrogenism of the polycystic ovary syndrome.

The 17,20 lyase activity lost to treatment with PP2A could be restored by re-phosphorylating P450c17 with endogenous kinases found in NCI-H295A cytoplasm. Thus one or more endogenous kinases and phosphatases appear to be in dynamic equilibrium, suggesting there may be multiple control points for the regulation of 17,20 lyase by P450c17 phosphorylation. It is likely that a cascade of other factors regulates both the positive action of the kinase and the negative action of the phosphatase. The demonstration that SET is present in NCI-H295A cells and can foster 17,20 lyase activity by inhibiting PP2A suggests that it is the first of these factors to be identified. Similarly, many protein kinases are themselves regulated by complex cascades of phosphorylation and dephosphorylation. Thus we propose that the regulation of the 17,20 lyase activity of P450c17 is positively regulated by a kinase pathway and negatively regulated by a phosphatase pathway, both of which contain multiple components, each of which represent a potential site of regulation (Fig. 8). We previously suggested that IGF-1 is associated with the induction of adrenarche (18) as disorders of insulin signal transduction appear to be associated with polycystic ovary syndrome; hence, we propose that the pathways regulating the phosphorylation and dephosphorylation of P450c17 are linked to the IGF-1 and insulin signal transduction pathways. Thus future elucidation of PP2A regulatory subunits and kinases involved in the P450c17 phosphorylation or dephosphorylation pathways should reveal candidate factors that may play key roles in polycystic ovary syndrome.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Proposal for the regulation of 17,20 lyase activity by reversible phosphorylation of P450c17. Arrows indicate stimulation, and blocked lines indicate inhibition. It is known that a cAMP-dependent serine/threonine (S/T) kinase stimulates the 17,20 lyase activity of P450c17 and a tyrosine (Y) kinase, and SET and okadaic acid (OA) inhibit PP2A. The nature of the kinases that regulate SET and that presumably regulate Ser/Thr kinases are unknown.

	ACKNOWLEDGEMENTS

We thank Dr. T. H. Tan (Baylor College, Houston, TX) for anti-PP4; Dr. K. Nagata (Tsukuba University, Tsukuba, Japan) for the anti-SET antibodies; Dr. M. R. Waterman (Vanderbilt University, Nashville, TN) for the pCwh17-mod(his)4; Dr. J. Chen (University of Illinois, Urbana) for plasmids pBJF and pBJF-Flag-PP2A; Dr. Stuart L. Schreiber (Harvard University, Cambridge, MA) for plasmids pBJF-Flag-PP4 and pBJF-Flag-PP6; Dr. J. W. M. Martens for help with the NCI-H295A cells; and Dr. C. E. Flück for preparing NCI-H295A RNA.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HD34449 (to W. L. M.), HD41958 (to W. L. M.), and HD27970 (to S. H. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pediatrics, Bldg. MR4, Rm. 209, University of California, San Francisco, CA 94143-0978. Tel.: 415-476-2598; Fax: 415-476-6286; E-mail: wlmlab@itsa.ucsf.edu.

Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M209527200

	ABBREVIATIONS

The abbreviations used are: DHEA, dehydroepiandrosterone; PP2A, protein phosphatase 2A; PP4, protein phosphatase 4; PP6, protein phosphatase 6; 17OH-Preg, 17OH-pregnonolone; 17OH-Prog, 17OH-progesterone; siRNA, small interfering RNA; DTT, dithiothreitol; RT, reverse transcriptase; Ni-NTA, nickel-nitrilotriacetic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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