Phosphorylation of Steroidogenic Acute Regulatory Protein (StAR) Modulates Its Steroidogenic Activity*

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR’s action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase.32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitrotranscription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [γ-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser 56 and Ser 194 ) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post-or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.
The recently discovered steroidogenic acute regulatory protein (StAR) 1 plays a critical role in the initial steps in steroidogenesis (1). Mutations in the StAR gene that inactivate the protein cause the most severe form of congenital adrenal hyperplasia in which the synthesis of all gonadal and adrenal steroids is markedly impaired (2,3). The StAR protein was originally identified by Orme-Johnson and colleagues (4 -7) and Stocco and associates (8 -11) as a 30-kDa phosphoprotein associated with mitochondria in gonadal and adrenal cells. Studies by Orme-Johnson and coworkers (5) first demonstrated a direct correlation between the appearance of a phosphorylated form of StAR and steroidogenesis. These authors suggested that StAR undergoes co-translational phosphorylation in response to cAMP, generating the active form of the protein.
The cDNAs encoding StAR have been determined for mouse (11), human (12), hamster (13), rat (14), cow (15), sheep (16), 2 and pig (17). Based on the comparison of the deduced amino acid sequences from these species and the predicted consensus motifs for protein kinase A (PKA) phosphorylation (R-R/K-X-S/T) (18), two conserved putative sites for PKA phosphorylation were identified (Fig. 1). These sites are serine 56/57 and serine 194/195 in the murine and human sequences, respectively. In the present study, we examined the roles of the serine 56/57 and serine 194/195 residues in StAR's steroidogenic activity using site-directed mutagenesis of these potential sites of cAMP-stimulated phosphorylation.

Construction of Mutant
StAR cDNA Constructs-The S57A, S195A, S195D, and S57A/S195A mutations were produced by site-directed mutagenesis using reagents purchased from CLONTECH. The wildtype and mutant human StAR cDNAs were cloned into pSV-SPORT-1. Each construct was sequenced to confirm the mutation(s) as described previously (19). The murine StAR cDNA constructs were prepared by Genosys (The Woodlands, TX). They were subcloned into the pCMV-5 eukaryotic expression vector.
Cell Culture and Evaluation of Steroidogenic Activity-COS-1 cells were cultured in 6-or 12-well plastic culture plates to 50 -80% confluence. The cells were transfected using 10 g/ml LipofectAMINE (Life Technologies, Inc.) with 1.0 g/ml of either an empty pSV-SPORT-1 plasmid, the wild-type, or mutant human StAR cDNAs in pSV-SPORT-1 with 1.0 g/ml of a plasmid-directing expression of a fusion protein consisting of P450scc, adrenodoxin and adrenodoxin reductase (1,3,20), kindly provided by Dr. Walter L. Miller, University of California, San Francisco. In studies of murine StAR, the corresponding pCMV-5-based plasmids were used. The culture media were changed after 24 h, and some cultures received 5 g/ml 22(R)-hydroxycholesterol. At the end of the treatment period, the media were collected for evaluation of steroidogenic activity, and the cells were scraped from the dishes in homogenization buffer consisting of 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 5 g/ml aprotinin (19). Relative steroidogenic activity was determined by normalizing pregnenolone production in the absence of 22(R)-hydroxycholesterol to pregnenolone formation in the presence of the exogenous substrate, which reflects maximal cholesterol side chain cleavage activity (3,19). Each experiment included triplicate cultures for each treatment group. Experiments were repeated on at least three separate occasions. Duncan's multiple range test was used to evaluate differences between control and mutated proteins.
Western Blotting-COS-1 cell extracts were harvested for Western blot analysis with an antibody raised against recombinant human StAR protein, to be described elsewhere (32), or antibody raised against a peptide sequence in murine StAR using previously published methods (11,19). The anti-recombinant human StAR antibody was used in studies of human StAR expression. The anti-murine StAR was used for studies of murine StAR expression. COS-1 cells collected into homogenization buffer were sonicated for 5 s. The disrupted cells were centrifuged at 600 ϫ g for 15 min and the resulting supernatant was used for the Western blot analysis as described previously (19).
Two-dimensional Gel Electrophoresis-Equal amount of protein (10 g) from each sample were loaded onto isoelectric focusing gels in capillary tubes prepared with ampholines with a pH range of 3 to 10. Two-dimensional SDS-PAGE was performed as described previously (21). The pH gradients of the isoelectric focusing gels were measured using two-dimensional standards obtained from Bio-Rad. After electrophoresis, the gels were transferred to nitrocellulose membranes for probing with anti-StAR antibody. In some cases, samples were pretreated with 1 IU of intestinal alkaline phosphatase (Boehringer Mannheim) for 30 min at 37°C prior to two-dimensional gel electrophoresis.
Incorporation of 32 P and 35 S and Immunoprecipitation of StAR Pro-tein-The COS-1 cells transfected with plasmid directing expression of wild-type and mutant StAR proteins were rinsed and incubated in phosphate-free Dulbecco's modified Eagle's medium without serum for 20 min and then labeled with [ 32 P]orthophosphate (200 Ci/ml). After labeling, cells were rinsed and preincubated with phosphate-buffered saline for 30 min, and then incubated with or without 1 mM 8-Br-cAMP in phosphate-buffered saline for 10 min at 37°C. The cells then were rapidly frozen in liquid nitrogen. For 35 S-labeling, cells were incubated in methionine-free medium containing [ 35 S]methionine/cysteine (100 Ci/ml) for 20 min. The radioactive medium was then replaced with culture medium containing unlabeled methionine and cysteine, and incubations were continued for 25 min to "chase" newly synthesized StAR preprotein into mature protein. The cells were rinsed and frozen as described above. The frozen cells were scraped into 500 l of ice-cold immunoprecipitation buffer consisting of 30 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 150 mM NaCl, 200 mM Na 2 V0 4 , 50 mM NaF, 5 g/ml aprotinin, 4 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride. After protein assay (Pierce), equal aliquots of protein from each cell extract were first incubated with 30 l of protein A-agarose (Life Technologies, Inc., 50% suspension) to eliminate proteins that nonspecifically bind to protein A in a total volume of 1 ml of immunoprecipitation buffer for 30 min at 4°C. After pelleting the protein A-agarose, supernatants were incubated with 10 l of anti-recombinant human StAR antibody and 30 l of protein A-agarose at 4°C for 3 h on a rocking platform. Immunocomplexes were washed four times by resuspension in 500 l of immunoprecipitation buffer and collected by centrifugation. The final pellets were resuspended in 50 l of SDS sample buffer (22) and then subjected to SDS-PAGE and autoradiography.
In Vitro Phosphorylation of StAR Proteins by PKA-Wild-type and mutant StAR proteins were synthesized, using an SP6 TNT-coupled in vitro transcription/translation kit following the manufacturer's protocol (Promega) for 2 h at 30°C. The in vitro translated proteins were incubated with 10 units of protein kinase A (PKA) catalytic subunit (Promega) in 10 mM HEPES buffer, pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, and 0.1 mM [␥-32 P]ATP in a total volume of 50 l. After a 30-min incubation at 30°C, the reaction was stopped by addition of SDS sample buffer. The samples were then subjected to immunoprecipitation and SDS-PAGE (22) followed by autoradiography as described above.

RESULTS
Human StAR Is Phosphorylated-Two-dimensional PAGE followed by Western blotting of extracts of COS-1 cells transfected with a human StAR expression plasmid demonstrated the presence of immunoreactive 30-kDa proteins, representing mature StAR, in four distinct forms ( Fig. 2A). Treatment of the COS-1 cell extracts with alkaline phosphatase prior to electrophoresis revealed that the more acidic immunoreactive proteins were sensitive to phosphatase treatment, suggesting that they are phosphoproteins (Fig. 2B). Since phosphatase treatment collapsed the four major immunoreactive proteins into two major spots, it appears that some other post-translational modification of StAR or phosphatase-insensitive modification accounts for the presence of at least one of the species. This finding is consistent with the earlier reports of Orme-Johnson and colleagues (4 -7) and Stocco and co-workers (8 -11). The presence of phosphorylated forms of StAR in the transfected COS-1 cells suggests that these hosts have the ability to generate these species in the basal state.
COS-1 cells expressing wild-type StAR were labeled with [ 32 P]orthophosphate overnight, and some cultures were treated with 1 mM 8-Br-cAMP for 10 min. Autoradiography performed on immunoprecipitated StAR proteins resolved by SDS-PAGE revealed incorporation of 32 P into both pre-and mature StAR proteins (Fig. 3). The labeling of StAR preprotein was significantly increased by the treatment with the cAMP analog, and the mobility of the preprotein from the cAMP-treated cell extracts was slightly retarded compared with the StAR preprotein in untreated cells. In addition, radiolabeled bands intermediate between the preprotein and 30-kDa mature protein were observed in the immunoprecipitates from 8-Br-cAMPtreated cells. This suggests sequential processing of the StAR preprotein as has been proposed by Clark et al. (11). Western blot analysis of the immunoprecipitates prepared from parallel cultures that were not labeled with 32 P also revealed retardation of the migration of the StAR preprotein extracted from the cAMP-treated COS-1 cells and the intermediate proteins between the pre-and mature StAR proteins (Fig. 3).
To identify which StAR residues are phosphorylated by PKA, we mutated the serine residues at codons 57 and 195, which are conserved potential PKA phosphorylation sites across all species, to alanines and analyzed patterns of phosphorylation of the mutants. In vitro transcription/translation was used to generate the wild-type and mutant proteins (Fig. 4A). Products of 37 and 32 kDa were identified, which represent the fulllength preprotein and initiation of translation of the preprotein from an internal methionine (19). Incubation of wild-type StAR preprotein with [␥-32 P]ATP documented enhanced 32 P incorporation into wild-type preprotein in the presence of PKA catalytic subunit (Fig. 4B). The incorporation of 32 P into StAR preproteins in the absence of added PKA catalytic subunit probably reflects the presence of protein kinase activity in the reticulocyte lysate in vitro transcription/translation system used to generate the proteins. We incubated equal amounts of the wild-type and mutant StAR preproteins with 32 P-labeled ATP and PKA catalytic subunit. As shown in Fig. 4C, wild-type human StAR was phosphorylated by PKA to greater extent than the S57A and S195A mutants. The S57A/S195A double mutant was phosphorylated to the same extent as wild-type protein incubated with [␥-32 P]ATP in the absence of PKA catalytic subunit.
We also transfected COS-1 cells with the wild-type and mutated StAR expression plasmids to compare the phosphorylation of mutant StAR proteins in the context of intact cells. After the COS-1 cells were labeled with [ 32 P]orthophosphate, they were stimulated with 1 mM 8-Br-cAMP for 10 min, and then cellular extracts were subjected to immunoprecipitation, SDS-PAGE, and autoradiography (Fig. 5). The incorporation of 32 P into the wild-type StAR preprotein was increased by 8-Br-cAMP treatment. The cAMP analog also provoked some increase in 32 P labeling of the S57A and S195A mutants, but not the double S57A/S195A mutant. cAMP treatment also retarded the mobility of the wild-type StAR preprotein, as noted above. In addition, 32 P incorporation into the mature StAR proteins was found in cells transfected with wild-type and S57A mutant, but not S195A and S57A/S195A double mutants.
When extracts of COS-1 cells transfected with the S195A StAR mutant were subjected to two-dimensional Western blot analysis, the pattern of immunoreactive proteins resembled that of the alkaline phosphatase-treated extracts of wild-type StAR transferase (Fig. 2C). These results suggest that 1) the wild-type StAR protein is phosphorylated by PKA, 2) that serine 57 and serine 195 are phosphorylated, 3) that cAMP increases phosphorylation of the StAR preprotein in COS-1 cells, and 4) that serine 195 is phosphorylated in the mature StAR protein, whereas serine 57 is either absent or not phosphorylated in mature StAR.
Steroidogenic Activity of StAR Phosphorylation Mutants-To examine the functional importance of phosphorylation of serine 57 and serine 195, we tested the steroidogenic activity of mutant proteins in which these serine residues were converted to alanines using COS-1 cells co-transfected with the cholesterol side chain cleavage system. Mutation of serine 195 resulted in an approximately 50% reduction (p Ͻ 0.001) in relative steroidogenic activity, whereas mutation of serine 57 had no significant effect on pregnenolone synthesis (Fig. 6A). The double mutation of S57A/S195A had no greater effect on steroidogenesis than the S195A mutation alone (the S57A/S195A mutant had a relative steroidogenic activity that was 45.2 Ϯ 6.7%, mean Ϯ S.E., of the activity of wild-type StAR). We prepared mutations of the corresponding serine residues in murine StAR and tested their steroidogenic activity. Conversion of serine 56 to an alanine had little effect on pregnenolone production, whereas mutation of serine 194 to an alanine residue reduced steroidogenesis by approximately 60% compared with wildtype murine StAR (p Ͻ 0.001) (Fig. 6C).
We next constructed a mutant human StAR cDNA in which serine 195 was converted into an aspartic acid residue to mimic the charge effect of phosphorylation at this site. The S195D mutant demonstrated a 20% increase in relative steroidogenic activity, which was not statistically significant (Fig. 6A). The mutations in the human and murine StAR proteins did not significantly alter the relative levels of StAR protein expressed in the transfected COS-1 cells as assessed by Western blotting (Fig. 6, B and D). To confirm that the S195A mutation did not interfere with protein expression we pulse-labeled transfected COS-1 cells with [ 35 S]methionine/[ 35 S]cysteine and immunoprecipitated the human wild-type and mutant StAR proteins following a 20-min chase period to permit newly synthesized StAR preproteins to be imported into mitochondria and processed. The immunoprecipitates were resolved by SDS-PAGE, and the preproteins and the mature proteins were visualized by autoradiography. For quantitation the corresponding areas of the gel were collected for liquid scintillation counting. Autoradiography demonstrated the presence of preprotein and mature protein in the wild-type and S195A transfectants. Label incorporation into the preproteins and mature proteins was not significantly different between the two transfectants (Table I). DISCUSSION Our observations demonstrate that human StAR protein is phosphorylated on serine residues at codons 57 and 195. These residues are in the context of consensus PKA phosphorylation sites, and these consensus sequences are conserved in the StAR proteins of all species studied to date. We have provided evidence that the phosphorylation of residues serine 57 and serine 195 is catalyzed by PKA and that incorporation of phosphate into these residues is increased by cAMP treatment of cells. However, it should be noted that the StAR protein may be phosphorylated at other sites and by other protein kinases including calcium/calmodulin-dependent protein kinase II (23), which phosphorylates serine or threonine residues in a context similar to that of PKA (18) and protein kinase C (7). StAR does not appear to be phosphorylated on tyrosine residues since we have not demonstrated a reaction of human or murine StAR with anti-phosphotyrosine antibodies. 3 Mutation of the serine at codon 195 in human StAR and the serine at codon 194 in murine StAR to nonphosphorylatable alanine residues reduces StAR's steroidogenic activity by approximately 50%, while mutation of serine 195 in human StAR to an aspartic acid residue slightly increases relative steroidogenic activity. Collectively, this evidence suggests that phosphorylation of serine 194/195 modulates StAR's functional activity. It is unlikely that the conversion of the serine 194/195 to an alanine diminished StAR's activity as a consequence of protein misfolding. First, the levels of expression of the S194A/S195A mutants were not significantly different from wild-type murine or human StAR or the S195D human StAR mutant. Second, the S195A mutant was labeled and processed to the mature protein similarly to wild-type StAR. Third, we have previously reported that mutation of the adjacent threonine at codon 196 in human StAR to an alanine does not affect steroidogenic activity, nor does mutation of serine 100 or serine 277 to alanines (24). Fourth, the S195A mutant StAR protein is localized to mitochondria by immunohistochemistry, just like wild-type StAR, and the S195A preprotein is as efficiently transported into isolated mitochondria and processed to mature protein in in vitro import assays as wild-type StAR preprotein. 4 The fact that the S56A/S57A mutation failed to significantly affect the steroidogenesis enhancing activity of the protein is not unexpected given our past observation that the first 62 amino-terminal residues of human StAR can be deleted without influencing steroidogenic activity (19). Because the S194A/S195A mutants retain 50% of the steroidogenic activity of wild-type StAR, we must conclude that in the context of the transfected cell system we used, phosphorylation of StAR at serine 194/195 is not absolutely required for the protein's function. 4 F. Arakane and J. F. Strauss, III, unpublished observations. FIG. 6. Relative steroidogenic activity of wild-type and phosphorylation site StAR mutants. A, COS-1 cells were transfected with the indicated human StAR plasmid and an expression plasmid for the human cholesterol side chain cleavage enzyme system. Relative steroidogenic activity assayed as pregnenolone production normalized to the conversion of 22(R)-hydroxycholesterol to pregnenolone is presented, taking the wild-type StAR value as 100%. Values are means Ϯ S.E. from four separate experiments. B, Western blot analysis was carried out on extracts of COS-1 cells to demonstrate expression of the human wild-type and mutant StAR proteins using antirecombinant human StAR antiserum. C, effects of mutation of serine residues 56 and 194 in murine StAR on pregnenolone synthesis. COS-1 cells were transfected with expression plasmids for murine wild-type StAR or the S56A or S194A mutants and pregnenolone secretion was determined. The results of a representative experimented replicated five times is presented. Values are presented with standard deviations. D, Western blot analysis of murine StAR proteins expressed in COS-1 cells using the anti-peptide antibody to murine StAR sequences.
Our findings are consistent with the notion that the activity of StAR can be increased by a co-or post-translational modification as originally suggested by Orme-Johnson and colleagues (5). StAR may be phosphorylated at serine residue 194/195 in response to an acute increase in cAMP levels and the subsequent activation of PKA. Since StAR has a short half-life, any change in the biological activity of newly synthesized StAR, as might result from phosphorylation of serine 194/195, would have a significant but short-lived effect on steroidogenesis. Thus, phosphorylation of StAR may be part of the mechanism of the immediate increase in steroid production following tropic stimulation of the adrenal cortex and gonads by ACTH and luteinizing hormone, respectively. cAMP also has long term effects on steroidogenesis by increasing the abundance of StAR mRNA and StAR protein, primarily through increases in the rate of transcription of the StAR gene (25,26). The exact mechanism by which phosphorylation changes the steroidogenic activity of StAR will remain a matter of speculation until the fundamental process by which StAR stimulates pregnenolone synthesis and the pathways that lead to its inactivation are elucidated.
Our two-dimensional Western blot studies indicate that COS-1 cells process StAR in a fashion that is similar to that previously reported in animal steroidogenic cells (4 -10). Several distinct charge isoforms were found and the more acidic species were sensitive to alkaline phosphatase treatment, consistent with the idea that they are derived from phosphorylation of the more basic forms of the protein. Despite this similarity, the COS-1 cell system has shortcomings that limit its utility for the study of the acute effects of cAMP on StAR function. Unlike normal steroidogenic cells, COS-1 cells co-transfected with the cholesterol side chain cleavage system and wild-type StAR do not respond with a statistically significant increase in pregnenolone production when challenged with cAMP. 3 This may reflect the fact that the expression plasmid floods the transfected cells with StAR protein, making it difficult to observe stimulatory actions of cAMPinduced protein phosphorylation, or that COS-1 cells lack important factors that are present in normal steroidogenic cells that are required for the acute steroidogenic response. Consequently, the effects of mutating serine 194/195 to an alanine or aspartic acid residue might be more profound in the context of a steroidsynthesizing cell responsive to tropic hormones.
What factors could be missing from the COS-1 cells to account for their inability to demonstrate a marked steroidogenic response to cAMP? The stimulation of pregnenolone production in the COS-1 cells by StAR may be limited by the availability of substrate. Indeed, the provision of an exogenous P450scc substrate in the form of a hydroxysterol increases pregnenolone synthesis by COS-1 cells about 2-fold above the levels produced from endogenous cholesterol in the presence of wild-type StAR (19). The rapid hydrolysis of stored cholesteryl esters in response to tropic stimulation probably contributes to the immediate increase in steroid synthesis by making available free cholesterol (1). This process of sterol ester mobilization may not take place in COS-1 cells. Alternatively, cAMP-induced modifications of other proteins that may be necessary for enhanced steroidogenesis, such as the peripheral benzodiazepine receptor (27), may not occur in COS-1 cells. Unfortunately, there are no known tropic hormone-responsive steroidogenic cells that lack StAR; so it is impossible for us to evaluate wild-type and mutant proteins in the ideal cell host.
In summary, the present findings demonstrate that serine 194/195, a potential site of phosphorylation mediated by PKA, is an important residue in StAR. The rapid stimulation of pregnenolone synthesis by cAMP has been well documented (28 -31), but the molecular basis of this response is poorly understood. Phosphorylation of the serine residue at codon 194/195 of StAR may account, in part, for the immediate increase in cholesterol side chain cleavage as a result of enhanced activity of the StAR protein. I Synthesis and processing of wild-type and S195A mutant StAR COS-1 cells were transfected with the indicated plasmid. Approximately 36 h after transfection the cells were placed into methionine-free medium and labeled for 20 min with [ 35 S]methionine/cysteine. After labeling, media were replaced with medium containing unlabeled methionine and cysteine, and incubations were continued for 25 min. Cell lysates were then prepared for immunoprecipitation of StAR. The immunoprecipitates were resolved by SDS-PAGE, and preproteins and mature proteins were visualized by autoradiography. The corresponding areas of the gel were excised for liquid scintillation counting. Values presented are means Ϯ S.E. from triplicate studies. Differences between means were not statistically significant (p Ͼ 0.05).