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Regulation of 17,20 Lyase Activity by Cytochrome b5 and by Serine Phosphorylation of P450c17

2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês

10.1074/jbc.m414673200

ISSN

1083-351X

Autores

Amit V. Pandey, Walter L. Miller,

Tópico(s)

Hormonal Regulation and Hypertension

Resumo

Cytochrome P450c17 catalyzes the 17α-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17α-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase. Cytochrome P450c17 catalyzes the 17α-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17α-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase. Cytochrome P450c17 is an essential steroid biosynthetic enzyme required for reproduction in all vertebrates. In mammals (1.Nakajin S. Hall P.F. Onoda M. J. Biol. Chem. 1981; 256: 6134-6139Abstract Full Text PDF PubMed Google Scholar, 2.Nakajin S. Shinoda M. Haniu M. Shively J.E. Hall P.F. J. Biol. Chem. 1984; 259: 3971-3976Abstract Full Text PDF PubMed Google Scholar, 3.Zuber M.X. Simpson E.R. Waterman M.R. Science. 1986; 234: 1258-1261Crossref PubMed Scopus (352) Google Scholar, 4.Lin D. Harikrishna J.A. Moore C.C.D. Jones K.L. Miller W.L. J. Biol. Chem. 1991; 266: 15992-15998Abstract Full Text PDF PubMed Google Scholar), fish (5.Sakai N. Tanaka M. Adachi S. Miller W.L. Nagahama Y. FEBS Lett. 1992; 301: 60-64Crossref PubMed Scopus (85) Google Scholar), birds (6.Nitta H. Osawa Y. Bahr J.M. Endocrinology. 1991; 129: 2033-2040Crossref PubMed Scopus (73) Google Scholar), and amphibians (7.Lutz L.B. Cole L.M. Gupta M.K. Kwist K.W. Auchus R.J. Hammes S.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13728-13733Crossref PubMed Scopus (146) Google Scholar), P450c17 catalyzes the steroid 17α-hydroxylase and 17,20 lyase activities needed to produce the 19-carbon precursors of sex steroids. The 17α-hydroxylase activity typically converts pregnenolone to 17α-hydroxypregnenolone (17OH-Preg) 1The abbreviations used are: 17OH-Preg, 17α-hydroxypregnenolone; 17OH-Prog, 17α-hydroxyprogesterone; DHEA, dehydroepiandrosterone; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilo triacetic acid; POR, P450 oxidoreductase; PP2A, protein phosphatase 2A; shRNA, small hairpin RNA; siRNA, small interfering RNA; IPTG, isopropyl-1-thio-β-d-galactopyranoside; HEK, human embryonic kidney; RT-PCR, reverse transcriptase-PCR; WT, wild type. and progesterone to 17α-hydroxyprogesterone (17OH-Prog), and the 17,20 lyase activity converts 17OH-Preg to dehydroepi- and rosterone (DHEA) and 17OH-Prog to androstenedione (Fig. 1). In human adrenal steroidogenesis, P450c17 is the qualitative regulator that determines the class of steroids synthesized in different cell types: in its absence, mineralocorticoids are produced; if only its 17α-hydroxylase activity is present, glucocorticoids are produced; if both its 17α-hydroxylase and 17,20 lyase activities are present, precursors of sex steroids are produced (8.Miller W.L. Auchus R.J. Geller D.H. Steroids. 1997; 62: 133-142Crossref PubMed Scopus (203) Google Scholar). The single human CYP17 gene (9.Picado-Leonard J. Miller W.L. DNA. 1987; 6: 439-448Crossref PubMed Scopus (390) Google Scholar) on chromosome 10q24 (10.Sparkes R.S. Klisak I. Miller W.L. DNA Cell Biol. 1991; 10: 359-365Crossref PubMed Scopus (127) Google Scholar) produces a single species of mRNA (11.Chung B.C. Picado-Leonard J. Haniu M. Bienkowski M. Hall P.F. Shively J.E. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 407-411Crossref PubMed Scopus (415) Google Scholar) encoding P450c17, which catalyzes both reactions on a single active site, apparently using the same chemical reaction mechanism (12.Auchus R.J. Miller W.L. Mol. Endocrinol. 1999; 13: 1169-1182Crossref PubMed Google Scholar). However, there are species-specific differences in these activities, especially concerning the 17,20 lyase reaction: the rodent and guinea pig enzymes use both 17OH-Preg and 17OH-Prog as substrates, the bovine enzyme mainly uses 17OH-Prog, and the human enzyme uses 17OH-Preg almost exclusively (1.Nakajin S. Hall P.F. Onoda M. J. Biol. Chem. 1981; 256: 6134-6139Abstract Full Text PDF PubMed Google Scholar, 2.Nakajin S. Shinoda M. Haniu M. Shively J.E. Hall P.F. J. Biol. Chem. 1984; 259: 3971-3976Abstract Full Text PDF PubMed Google Scholar, 4.Lin D. Harikrishna J.A. Moore C.C.D. Jones K.L. Miller W.L. J. Biol. Chem. 1991; 266: 15992-15998Abstract Full Text PDF PubMed Google Scholar, 13.Auchus R.J. Lee T.C. Miller W.L. J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 14.Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar). Similar to all other microsomal forms of cytochrome P450, the catalytic activities of P450c17 require electron donation from NADPH through the intermediacy of a membrane-bound flavoprotein termed P450 oxidoreductase (POR) (15.Lu A.Y. Junk K.W. Coon M.J. J. Biol. Chem. 1969; 244: 3714-3721Abstract Full Text PDF PubMed Google Scholar, 16.Shen A.L. Kasper C.B. J. Biol. Chem. 1995; 270: 27475-27480Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 17.Flück C.E. Tajima T. Pandey A.V. Arlt W. Okuhara K. Verge C.F. Jabs E.W. Mendonca B.B. Fujieda K. Miller W.L. Nat. Genet. 2004; 36: 228-230Crossref PubMed Scopus (407) Google Scholar). All cytochrome P450 enzymes catalyze multiple reactions, but the ratios of these multiple reactions are determined primarily by substrate concentrations. By contrast, P450c17 appears to be unique in that the ratio of its two principal activities is developmentally regulated. Human serum concentrations of cortisol, an index of 17α-hydroxylase activity, remain constant as a function of age, but concentrations of DHEA, an index of 17,20 lyase activity, rise over 100-fold during adrenarche (18.Orentreich N. Brind J.L. Rizer R.L. Vogelman J.H. J. Clin. Endocrinol. Metab. 1984; 59: 551-555Crossref PubMed Scopus (1271) Google Scholar), an event that is contemporaneous with, but independent of puberty (19.Apter D. Pakarinen A. Hammond G.L. Vihko R. Acta Paediatr. Scand. 1979; 68: 599-604Crossref PubMed Scopus (84) Google Scholar, 20.Sklar C.A. Kaplan S.L. Grumbach M.M. J. Clin. Endocrinol. Metab. 1980; 51: 548-556Crossref PubMed Scopus (212) Google Scholar, 21.Miller W.L. Acta Paediatr. Suppl. 1999; 88: 60-66Crossref PubMed Google Scholar, 22.Arlt W. Martens J.W. Song M. Wang J.T. Auchus R.J. Miller W.L. Endocrinology. 2002; 143: 4665-4672Crossref PubMed Scopus (88) Google Scholar). The ratio of 17,20 lyase activity to 17α-hydroxylase activity can be regulated by three distinct post-translational mechanisms. First, the presence of high molar ratios of POR to P450c17 favor the 17,20 lyase reaction (14.Lin D. Black S.M. Nagahama Y. Miller W.L. Endocrinology. 1993; 132: 2498-2506Crossref PubMed Google Scholar, 23.Yanagibashi K. Hall P.F. J. Biol. Chem. 1986; 261: 8429-8433Abstract Full Text PDF PubMed Google Scholar). Second, 17,20 lyase activity can be enhanced by the presence of cytochrome b5 (24.Onoda M. Hall P.F. Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (118) Google Scholar, 25.Ishii-Ohba H. Matsumura R. Inano H. Tamaoki B. J. Biochem. (Tokyo). 1984; 95: 335-343Crossref PubMed Scopus (18) Google Scholar, 26.Katagiri M. Kagawa N. Waterman M.R. Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (212) Google Scholar), which acts allosterically to foster interactions with POR (13.Auchus R.J. Lee T.C. Miller W.L. J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 27.Geller D.H. Auchus R.J. Miller W.L. Mol. Endocrinol. 1999; 13: 167-175Crossref PubMed Scopus (141) Google Scholar). Third, serine/threonine phosphorylation of P450c17 increases 17,20 lyase activity but does not affect 17α-hydroxylase activity (28.Zhang L.H. Rodriguez H. Ohno S. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10619-10623Crossref PubMed Scopus (406) Google Scholar, 29.Pandey A.V. Mellon S.H. Miller W.L. J. Biol. Chem. 2003; 278: 2837-2844Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In some P450-mediated drug metabolism reactions, cytochrome b5 appears to act as an alternative electron donor that can substitute for POR in the donation of second electron in the P450 cycle (30.Yamazaki H. Gillam E.M. Dong M.S. Johnson W.W. Guengerich F.P. Shimada T. Arch. Biochem. Biophys. 1997; 342: 329-337Crossref PubMed Scopus (130) Google Scholar, 31.Guryev O.L. Gilep A.A. Usanov S.A. Estabrook R.W. Biochemistry. 2001; 40: 5018-5031Crossref PubMed Scopus (81) Google Scholar, 32.Yamazaki H. Shimada T. Martin M.V. Guengerich F.P. J. Biol. Chem. 2001; 276: 30885-30891Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) but it does not function in this fashion to foster 17,20 lyase activity, as apo b5, which is devoid of heme, is as effective as holo b5 (13.Auchus R.J. Lee T.C. Miller W.L. J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 32.Yamazaki H. Shimada T. Martin M.V. Guengerich F.P. J. Biol. Chem. 2001; 276: 30885-30891Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Mutations in the POR binding site of P450c17 selectively reduce 17,20 lyase activity (33.Geller D.H. Auchus R.J. Mendonca B.B. Miller W.L. Nat. Genet. 1997; 17: 201-205Crossref PubMed Scopus (261) Google Scholar); when cytochrome b5 is added, 17,20 lyase activity is partially restored, increasing the Vmax but not influencing the Km of the mutants (27.Geller D.H. Auchus R.J. Miller W.L. Mol. Endocrinol. 1999; 13: 167-175Crossref PubMed Scopus (141) Google Scholar), thus confirming the allosteric action of cytochrome b5. The mechanism by which P450c17 phosphorylation augments 17,20 lyase activity is not known, nor is it known whether the state of P450c17 phosphorylation influences the allosteric action of cytochrome b5. Using purified, catalytically active, bacterially-expressed human P450c17, POR, and b5 in vitro and RNA interference knockdown of cytochrome b5 in human adrenal NCI-H295A cells, we now show that these two mechanisms of augmenting 17,20 lyase activity function independently. Cell Culture and Microsome Preparation—NCI-H295A cells (34.Rodriguez H. Hum D.W. Staels B. Miller W.L. J. Clin. Endocrinol. Metab. 1997; 82: 365-371Crossref PubMed Scopus (89) Google Scholar), an adherent population of human adrenocortical carcinoma NCI-H295 cells (35.Gazdar A.F. Oie H.K. Shackleton C.H. Chen T.R. Triche T.J. Myers C.E. Chrousos G.P. Brennan M.F. Stein C.A. La Rocca R.V. Cancer Res. 1990; 50: 5488-5496PubMed Google Scholar, 36.Staels B. Hum D.W. Miller W.L. Mol. Endocrinol. 1993; 7: 423-433Crossref PubMed Scopus (176) Google Scholar), were grown in 150-mm Petri dishes as described (34.Rodriguez H. Hum D.W. Staels B. Miller W.L. J. Clin. Endocrinol. Metab. 1997; 82: 365-371Crossref PubMed Scopus (89) Google Scholar). HepG2, JEG3 (37.Wijesuriya S.D. Zhang G. Dardis A. Miller W.L. J. Biol. Chem. 1999; 274: 38097-38106Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and HEK293 (38.Louis N. Evelegh C. Graham F.L. Virology. 1997; 233: 423-429Crossref PubMed Scopus (158) Google Scholar) cells were grown as described. NCI-H295A cells from several plates were collected by scraping, washed with chilled phosphate-buffered saline (PBS), suspended in 50 mm sodium phosphate containing 150 mm KCl, and lysed by sonication. Unbroken cells and mitochondria were removed by centrifugation at 10,000 × g for 15 min, and microsomes were collected by ultracentrifugation at 100,000 × g for 90 min in a Beckman T-100 rotor. Microsomes were resuspended in 50 mm potassium phosphate buffer containing 20% glycerol. Cytochrome b5 Expression Analysis—Expression of cytochrome b5 in various cell lines was analyzed by RT-PCR using GAPDH as control. The primer sequences used were for cytochrome b5: sense, 5′-ATGGCAGAGCAGTCGGACGA-3′ and antisense, 5′-TCAGTCCTCTGCCATGTATAG-3′; for OMb5: sense, 5′-ATGGCGACTGCGGAAGCTA-3′ and antisense, 5′-TCAGGAGGATTTGCTTTCCGA-3′; for GAPDH: sense, 5′-GTATCGTGGAAGGACTCAT-3′ and antisense, 5′-TACTCCTTGGTGGCCATGT-3′. All PCR reactions were performed for 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C 45s followed by 1 cycle of 72 °C for 10 min, and products were separated on 10% Tris borate/EDTA acrylamide gel, stained with ethidium bromide and visualized on a Kodak EDS 290 gel documentation system. Expression and Purification of Human P450c17—Human P450c17 was expressed in Escherichia coli and purified as described (39.Imai T. Globerman H. Gertner J.M. Kagawa N. Waterman M.R. J. Biol. Chem. 1993; 268: 19681-19689Abstract Full Text PDF PubMed Google Scholar). The plasmid pCWH17-mod(His)4 containing the cDNA for N-terminally modified human P450c17 (40.Brock B.J. Waterman M.R. Biochemistry. 1999; 38: 1598-1606Crossref PubMed Scopus (115) Google Scholar) was transformed into E. coli JM109, colonies were selected on LB agar plates containing 100 μg/ml carbenicillin, and a single colony was grown to A600 of 0.4–0.6 in LB broth containing 100 μg/ml carbenicillin. From this culture, 1 ml of bacteria were seeded into terrific broth containing 40 μm FeCl3,4 μm ZnCl2,2 μm CoCl2, 2 μm Na2MoO4, 2 μm CaCl2, 2 μm CuCl2, 2 μm H3BO3, and 10% v/v potassium phosphate solution (0.17 m KH2PO4, 0.72 m K2HPO4, pH 7.4) and 100 μg/ml carbenicillin and grown at 30 °C with shaking at 250 rpm. At A600 of 0.5–0.7, 0.4 mm isopropyl-1-thio-β-d-galactopyranoside (IPTG) and 0.4 mm δ-amino levulinic acid were added, and the culture was shaken at 125 rpm for 36 h at 30 °C. The bacteria were then chilled on ice, pelleted by centrifugation at 5000 × g for 10 min at 4 °C, washed once with PBS, and resuspended in 100 mm Tris-HCl, pH 7.8 containing 500 mm sucrose and 0.5 mm EDTA (10 ml/g of pellet). Lysozyme (0.1 mg/ml) was added to the bacterial suspension, and the cells were kept on ice for 30 min with occasional stirring. Spheroplasts were harvested by centrifugation at 12,000 × g for 15 min at 4 °C and resuspended in 100 mm potassium phosphate pH 7.6 containing 6 mm magnesium acetate, 20% glycerol, and 0.1 mm phenylmethylsulfonyl fluoride. The resulting spheroplasts were sonicated on ice with 15–20 cycles of 20-s pulses followed by 30 s cooling at 50% power using a Fisher scientific 550 sonicator with a microprobe. The lysate was cleared of unbroken cells and debris by centrifugation at 10,000 × g for 10 min at 4 °C, and membranes were pelleted by ultracentrifugation in a Beckman T-100 rotor at 100,000 × g for 90 min. The resultant pellet containing human P450c17 was used for enzyme assays and purification of P450c17; cytochrome P450 content was measured by reduced carbon monoxide binding spectra (41.Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar). P450c17 was purified from the E. coli membranes by solubilization in 0.7% Triton X-114 followed by chromatography on Ni-NTA and hydrophobic interaction chromatography as described (29.Pandey A.V. Mellon S.H. Miller W.L. J. Biol. Chem. 2003; 278: 2837-2844Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 39.Imai T. Globerman H. Gertner J.M. Kagawa N. Waterman M.R. J. Biol. Chem. 1993; 268: 19681-19689Abstract Full Text PDF PubMed Google Scholar). Purified P450c17 was resuspended in 100 mm potassium phosphate, pH 7.4, containing 20% glycerol and 0.1% Triton X-100. Phosphatase Treatment of Bacterially Expressed Human P450c17— Purified preparations of P450c17 (5 μg) were treated with 5 units of alkaline phosphatase or protein phosphatase 2A (PP2A) for 15 min at 25 °C, and the reaction was stopped by adding 1 mm sodium orthovanadate, 50 mm NaF, and/or 50 μm okadaic acid (Calbiochem) in the case of protein PP2A. P450c17 treated with phosphatases was repurified on a Ni-NTA spin column (Amersham Biosciences) and used for enzyme assays. The amount of phosphate released into the supernatant was monitored by malachite green reaction (BioMol Laboratories). Expression and Purification of Human POR—Human POR was expressed in bacteria by transforming E. coli BL21(DE3)pLysS cells with a pET22b vector (Novagen) containing human POR cDNA (17.Flück C.E. Tajima T. Pandey A.V. Arlt W. Okuhara K. Verge C.F. Jabs E.W. Mendonca B.B. Fujieda K. Miller W.L. Nat. Genet. 2004; 36: 228-230Crossref PubMed Scopus (407) Google Scholar). Freshly transformed E. coli were selected on a LB agar plate with 100 μg/ml carbenicillin and 34 μg/ml chloramphenicol, and a single colony was seeded into LB media containing 100 μg/ml carbenicillin and 34 μg/ml chloramphenicol and grown at 28 °C to A600 of about 0.5. From this culture, 1 ml of bacteria were seeded into 150 ml of terrific broth supplemented with 40 μm FeCl3, 4 μm ZnCl2, 2 μm CoCl2, 2 μm Na2MoO4, 2 μm CaCl2, 2 μm CuCl2, 2 μm H3BO3, and 10% v/v potassium phosphate solution (0.17 m KH2PO4, 0.72 m K2HPO4, pH 7.4), 100 μg/ml carbenicillin and 34 μg/ml chloramphenicol and grown at 30 °C to A600 of 0.4–0.7, 0.4 mm IPTG and 0.1 mg/ml riboflavin were added, and the bacteria were shaken at 125 rpm for another 16 h before harvesting by centrifugation at 5000 × g for 10 min at 4 °C. The bacteria were then washed with PBS, treated with lysozyme (0.1 mg/ml) and EDTA (0.1 mm, pH 8.0), and membranes were prepared as described for P450c17. Membranes were dissolved in 100 mm phosphate buffer containing 100 mm NaCl, 1.5% Triton X-100 and 0.1 mm phenylmethylsulfonyl fluoride, and POR was purified by affinity chromatography on 2′-5′ ADP-agarose as described (42.Smith G.C. Tew D.G. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8710-8714Crossref PubMed Scopus (145) Google Scholar). The catalytic activity of this POR preparation was assessed by assays using either cytochrome c or NADPH as substrates as described (17.Flück C.E. Tajima T. Pandey A.V. Arlt W. Okuhara K. Verge C.F. Jabs E.W. Mendonca B.B. Fujieda K. Miller W.L. Nat. Genet. 2004; 36: 228-230Crossref PubMed Scopus (407) Google Scholar). Expression and Purification of Cytochrome b5—Human cytochrome b5 was prepared in E. coli strain BL21(DE3) transformed with plasmid pLW01b5 containing the cDNA for human cytochrome b5 and selected over several cycles, as described (43.Miroux B. Walker J.E. J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1586) Google Scholar). A single colony of transformed E. coli was inoculated into LB media containing 100 μg/ml carbenicillin, grown to an A260 of 0.5–0.7, and IPTG was added to a final concentration of 0.4 mm. Cell density was measured every 30 min; when the A600 nm started to rise again (90–180-min postinduction), 100 μl of cells diluted 1:50 were plated on LB agar plates containing 100 μg/ml carbenicillin and 0.4 mm IPTG and allowed to grow overnight at 37 °C. A single colony was grown the next day and the process of induction and selection with carbenicillin and IPTG was repeated three more times. After four cycles of selection, the E. coli were transformed with plasmid pLysS Rare, which encodes lysozyme and six rare tRNAs that assist the expression of mammalian proteins by complementing the codon bias of E. coli. The resultant E. coli were used for expression of cytochrome b5. Expression and purification was performed as described previously (44.Mulrooney S.B. Waskell L. Protein. Expr. Purif. 2000; 19: 173-178Crossref PubMed Scopus (68) Google Scholar). Suppression of Cytochrome b5 by Short Hairpin RNA (shRNA) in NCI-H295A Cells—NCI-H295A cells were transfected with the retroviral vector pSUPERretro (45.Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar, 46.Brummelkamp T.R. Bernards R. Agami R. Cancer Cell. 2002; 2: 243-247Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar) expressing shRNA targeted against both types 1 and 2 human cytochrome b5. The pSUPERretro vector was digested with BglI and HindIII and ligated to the 64-mer oligonucleotide duplexes (sense 5′-GATCCCCCAAGCTGGAGGTGACGCTATTCAAGAGATAGCGTCACCTCCAGCTTGTTTTTGGAAA-3′and antisense 3′-GGGGTTCGACCTCCACTGCGATAAGTTCTCTATCGCAGTGGAGGTCGAACAAAAACCTTTTCGA-5′) encoding small interfering RNA (siRNA) against cytochrome b5 and a 9-nucleotide loop region. The recombinant plasmid was purified from bacterial cultures and digested with EcoRI and HindIII to check the inserts. Purified plasmid was transformed into NCI-H295A, and cells containing the virus were selected by puromycin and used for cytochrome b5 measurement and P450c17 enzyme assays. Measurement of Cytochrome b5 Protein and mRNA—The cytochrome b5 content of NCI-H295A cells was measured by differential spectroscopy (47.Strittmatter P. Velick S.F. J. Biol. Chem. 1956; 221: 253-264Abstract Full Text PDF PubMed Google Scholar). Cells were homogenized in sodium phosphate buffer (50 mm, pH 7.4) containing 150 mm KCl; the homogenate was cleared by centrifugation at 5000 × g for 10 min, placed into reference and sample cuvettes and baseline spectra were recorded between 400 and 500 nm at a speed of 120 nm/min. Cytochrome b5 in the sample cuvette was reduced with 1 mg sodium dithionite, and the spectra were recorded again. Cytochrome b5 content was estimated by the difference in absorbance at 423 and 490 nm using a millimolar extinction coefficient of 181 mmol cm–1 (47.Strittmatter P. Velick S.F. J. Biol. Chem. 1956; 221: 253-264Abstract Full Text PDF PubMed Google Scholar). Cytochrome b5 mRNA was estimated by RT-PCR. In Vitro Phosphorylation of P450c17—To prepare a cytoplasmic fraction enriched for the kinases that phosphorylate P450c17 and devoid of phosphatase activity, NCI-H295A cells were lysed, and cytosol was passed over a γ-ATP-Sepharose column (Upstate Biotechnologies); the column was washed with NAD, NADP, ADP, and AMP, and ATP-binding proteins were eluted with 10 mm ATP (29.Pandey A.V. Mellon S.H. Miller W.L. J. Biol. Chem. 2003; 278: 2837-2844Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Kinase activity was checked by phosphorylation of microsomes or purified bacterially expressed recombinant human P450c17 by incorporation of [γ-32P]ATP. Purified P450c17 was phosphorylated in vitro using 6 μg of purified P450c17 incubated with 10 μg of kinase fraction in the presence of 50 mm HEPES buffer, 10 mm ATP, 60 mm MgCl2, and 10 μm okadaic acid at 25 °C for 30 min and phosphorylated P450c17 was purified on mini Ni-NTA columns (29.Pandey A.V. Mellon S.H. Miller W.L. J. Biol. Chem. 2003; 278: 2837-2844Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). P450c17 Enzyme Assays—The 17α-hydroxylase and 17,20 lyase activities of P450c17 were assayed as described (13.Auchus R.J. Lee T.C. Miller W.L. J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 39.Imai T. Globerman H. Gertner J.M. Kagawa N. Waterman M.R. J. Biol. Chem. 1993; 268: 19681-19689Abstract Full Text PDF PubMed Google Scholar). Purified P450c17 (10 pmol) and POR (20 pmol) were incubated with 100 mm potassium phosphate, 6 mm potassium acetate, 10 mm MgCl2, 1 mm reduced glutathione, 20% glycerol, 20 μg phosphatidylcholine, 3 units of glucose-6-phosphate dehydrogenase, 0.1 mm glucose-6-phosphate and radiolabeled substrate ([14C]progesterone for hydroxylase assay and [3H]17OH-Preg for lyase assay) with or without 10 pmol of cytochrome b5 for 5 min at 25 °C in a total volume of 200 μl. The reaction was started by addition of 20 μl of 10 mm NADPH and incubations at 37 °C were carried out for various times and stopped by adding ethyl acetate/iso-octane (3:1) to extract the steroids. Steroids from different reactions were spotted on silica gel 60 F-254 thin layer chromatography plates (Merck) and developed with ethyl acetate/chloroform (3:1) (4.Lin D. Harikrishna J.A. Moore C.C.D. Jones K.L. Miller W.L. J. Biol. Chem. 1991; 266: 15992-15998Abstract Full Text PDF PubMed Google Scholar). Plates were dried, and steroids were quantitated by autoradiography on a Storm 860 phosphorimager using Image Quant software. Kinetic behavior was approximated as a Michaelis-Menten system. Curve fitting and calculations of maximum velocity (Vmax) and apparent Michaelis constant (Km) values were performed using LEONORA (48.Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1995Google Scholar). Isoforms of Cytochrome b5 in NCI-H295A Cells—Three forms of cytochrome b5 have been described: the 98-amino acid soluble and 134-amino acid microsomal form are encoded by one gene (49.Giordano S.J. Steggles A.W. Biochem. Biophys. Res. Commun. 1991; 178: 38-44Crossref PubMed Scopus (35) Google Scholar, 50.Giordano S.J. Yoo M. Ward D.C. Bhatt M. Overhauser J. Steggles A.W. Hum. Genet. 1993; 92: 615-618Crossref PubMed Scopus (8) Google Scholar) and a 146-amino acid form associated with the outer mitochondrial membrane (OMb5) is encoded by a second gene (51.Rivera M. Barillas-Mury C. Christensen K.A. Little J.W. Wells M.A. Walker F.A. Biochemistry. 1992; 31: 12233-12240Crossref PubMed Scopus (84) Google Scholar, 52.Kuroda R. Ikenoue T. Honsho M. Tsujimoto S. Mitoma J.Y. Ito A. J. Biol. Chem. 1998; 273: 31097-31102Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 53.Altuve A. Silchenko S. Lee K.H. Kuczera K. Terzyan S. Zhang X. Benson D.R. Rivera M. Biochemistry. 2001; 40: 9469-9483Crossref PubMed Scopus (55) Google Scholar). To identify the forms of cytochrome b5 found in NCI-H295A cells, we performed RT-PCR with two pairs of oligonucleotide primers that will amplify the three products of the two genes. In human liver HepG2 cells, the mRNAs for microsomal cytochrome b5 and for OMb5 were found in approximately equal amounts, but in human kidney HEK293, human placenta JEG-3 cells, and especially in human adrenal NCI-H295A cells, the mRNA for microsomal cytochrome b5 was much more abundant than the mRNA for OMb5 (Fig. 2A). The mRNA for the soluble form, generally associated with erythropoietic tissues, was not detected in the cell lines tested. As human P450c17 is a microsomal enzyme and as the mRNA for the microsomal form of cytochrome b5 was most abundant in adrenal cells, we focused attention on microsomal cytochrome b5. Knockdown of Cytochrome b5 in NCI-H295A Cells—To determine the effect of cytochrome b5 on the catalytic activities of P450c17 in steroidogenic cells, we used RNA interference to knock down cytochrome b5 mRNA in human adrenal NCI-H295A cells (Fig. 2B). An shRNA targeted against cytochrome b5 was transfected into human adrenal NCI-H295A cells, and cells were harvested 24, 48, 72, and 96 h later. Expression of this shRNA caused a gradual decrease in spectrally assayable cytochrome b5 over time: cytochrome b5 was reduced by 15% after 24 h, 65% after 48 h, and 85% after 72 h (Fig. 2C). Knockdown of cytochrome b5 had no effect on 17α-hydroxylase activity in NCI-H295A cells for up to 96 h (Fig. 2, D and E). There was no measurable change in the 17,20 lyase activity in the first 24 h, but 17,20 lyase activity was reduced 15% after 48 h and 30% after 72–96 h (Fig. 2, D and E). This decrease in 17,20 lyase activity was much less than the decrease in cytochrome b5 protein, suggesting that another factor was promoting 17,20 lyase activity. Increased Phosphorylation of P450c17 Counters the Effect of Reduced Cytochrome b5 on 17,20 Lyase Activity—Because reducing cytochrome b5 in intact cells had only a modest effect on 17,20 lyase activity, we sought to examine the potential interactions between the presence of cytochrome b5 and the phosphorylation of P450c17 on 17,20 lyase activity. Okadaic acid, an inhibitor of serine/threonine phosphatases, specifically increases 17,20 lyase activity in NCI-H295A cells and increases P450c17 phosphorylation (29.Pandey A.V. Mellon S.H. Miller W.L. J. Biol. Chem. 2003; 278: 2837-2844Abstract Full Text Full Text PDF PubMed Scopus (122) Goo

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