Phosphorylation of an N-terminal Motif Enhances DNA-binding Activity of the Human SRY Protein*

Marion DesclozeauxDagger , Francis PoulatDagger , Pascal de Santa Barbara, Jean-Paul Capony, Patric Turowski, Philippe Jay, Catherine Méjean, Brigitte Moniot, Brigitte Boizet, and Philippe Berta§

From the Centre de Recherche de Biochimie Macromoléculaire, ERS155 CNRS, 1919 route de Mende, BP 5051, 34033 Montpellier Cedex, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Of the several strategies that eukaryotes have evolved to modulate transcription factor activity, phosphorylation is regarded as one of the major mechanisms in signal-dependent transcriptional control. To conclusively demonstrate that the human sex-determining gene SRY is affected by such a post-translational control mechanism, we have analyzed its phosphorylation status in living cells. In the present study, we show that the cyclic AMP-dependent protein kinase (PKA) phosphorylates the human SRY protein in vitro as well as in vivo on serine residues located in the N-terminal part of the protein. This phosphorylation event was shown to positively regulate SRY DNA-binding activity and to enhance the ability of SRY to inhibit a basal promoter activity located downstream of an SRY DNA-binding site concatamer. Together these results strongly support the hypothesis that human SRY is a natural substrate for PKA in vivo and that this phosphorylation significantly modulates its major activity, DNA-binding, thereby possibly altering its biological function.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In eutherian mammals, testis determination is under the control of the Y chromosome which commits the bipotential embryonic gonad to the testicular pathway. The initial step of this process is the differentiation of pre-Sertoli into Sertoli cells that will next produce anti-Müllerian hormone and will direct the other gonadal cell lineages toward the testicular pathway (1).

In 1990, the SRY (sex determining region of Y) gene was isolated from the human Y chromosome (2) and was subsequently shown to equate with the elusive testis determining factor (TDF)1 (3, 4). Sequence analysis of SRY revealed that the protein it encodes is a member of a large family of nuclear proteins harboring a 79-amino acid motif known as an HMG (high mobility group) box (5). More precisely, the SRY protein belongs to a subclass of HMG box-containing proteins that bind specifically to DNA sequences. This subclass includes transcriptional regulators such as TCF1 (6) and LEF-1 (7, 8), or diverse SOX (SRY-box related) proteins (9).

In vitro binding assays show that the SRY HMG box exhibits sequence-specific DNA-binding to the consensus sequence AACAAT (10) involving interaction in the DNA minor groove (11). In addition, the SRY protein was shown to be nuclear (12) and to induce a strong bending of the DNA target (13). All these characteristics are consistent with a role for SRY in the regulation of transcription. The functional specificity of SRY with respect to other SOX proteins (defined by the presence of an SRY box), cannot be attributable to differences in their DNA-binding properties alone (9), but probably results from interactions with other proteins. The lack of a potential trans-regulation domain in the human SRY protein sequence led to diverse speculations about its mode of action (14). The strong bending of DNA, induced by SRY, could result in the assembly of multiple proteins/DNA complexes as reported for other HMG-box containing proteins (15, 16), and the search for SRY-interacting proteins involved in such multiple complexes has been undertaken in some laboratories. If this hypothesis is correct, target gene specificity would then be the result of protein-protein interactions in addition to protein-DNA interactions. Consistent with this is the recent cloning of SIP-1, a PDZ motif containing protein which interacts with the C-terminal domain of SRY (17), or the demonstration of potential SRY-calmodulin binding (18).

To date, no post-translational modifications of the SRY protein have been reported. Since many diverse stimuli that affect gene expression during cell differentiation also lead to protein kinase activation, it is tempting to hypothesize a direct regulation of SRY function by phosphorylation. Such modulation of transcription factor activity by phosphorylation is well documented and is known to play a pivotal role in the regulation of various transcription activities (for a review, see Ref.19). Phosphorylation can affect either the subcellular localization of the transcription factor (20, 21), its transactivation potential (22, 23), its DNA-binding activity (24, 25), or all of these processes.

To further dissect the mechanisms whereby human SRY induces Sertoli cell determination, we have investigated whether SRY activity can be modulated via phosphorylation. We show that SRY is phosphorylated in vitro as well as in vivo at a protein kinase A (PKA) motif located in the N-terminal portion. This phosphorylation status is enhanced when using a PKA activator. Using an electrophoretic mobility shift assay, we demonstrate that this phosphorylation enhances the DNA-binding activity of the SRY protein. In contrast, studies with mutant SRY protein show that PKA phosphorylation failed to significantly alter SRY subcellular localization. We have also addressed the regulation of SRY in vivo by examining the basal transcription activity of a thymidine kinase promoter with multiple upstream SRY DNA-binding sites. Using this assay, we have demonstrated that PKA phosphorylation of the SRY protein correlates well with an increase of its repressor activity on such a construct.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture and Transfection Assays-- The human NT2/D1 cells (N-Tera 2, clone D1, a human pluripotent embryonic carcinoma cell line, ATCC No. CRL 1973) were obtained from the American Type Culture Collection (ATCC, Biovaley, France). NT2/D1 and COS7 cells were cultured in Dulbecco's modified Eagle's medium (Imperial Laboratories, Flobio, France) containing 10% (v/v) fetal calf serum (Life Technologies Inc.), penicillin/streptomycin, and 2 mM glutamine with 5% pCO2 at 37 °C in humidified air. Plasmids used for transfection were purified using the Maxiprep reagent system (Qiagen). COS7 cells at 60-80% confluence were washed twice with serum-free medium before adding 1 µg of reporter plasmid and 500 ng of SRY (wild-type or mutants) pJ3Omega expression plasmid (26) with 7 µl of LipofectAMINE (Life Technologies Inc.) in 200 µl of serum-free medium. After 6 h of incubation, the medium was replaced with 2 ml of medium supplemented with 10% serum, and cells were harvested after 48 h of culture. CAT assays were performed on cell extracts using 1-deoxy-(dichloro-acetyl-1-3H)chloramphenicol (200 mCi/mmol, Amersham, UK) by nonchromatographic method as described (27).

Phosphoamino Acid Analysis-- Radiolabeled immunoprecipitated SRY was separated by SDS-PAGE (15% acrylamide) and electrophoretically transferred to Immobilon-P membrane (Millipore). The band corresponding to SRY (27 kDa) was individualy excised from the Immobilon membrane, hydrolyzed in N HCl for 90 min at 110 °C, and subjected to phosphoamino acid analysis as described (28). The hydrolyzed sample was mixed with 1 µg each of unlabeled phosphoserine, phosphothreonine, and phosphotyrosine as standards. The sample was next spotted onto a thin layer chromatography plate and separated by two-dimensional high voltage electrophoresis. Amino acid standards were visualized with ninhydrin and 32P comigrating with the standards detected by autoradiography.

Production and Purification of Bacterially Expressed pGex-SRY Fusion Protein and Mutagenesis-- Because of the low level of full-length SRY protein production using diverse expression systems, the first 100 5'-nucleotides of the human SRY cDNA were replaced by synthetic DNA, constructed to accord with bacterial codon usage. The reconstructed SRY was then cloned in the pGex4T1 expression vector. The recombinant protein was produced in the bacterial strain BL21(DE3) after induction by isopropyl-1-thio-beta -D-galactopyranoside (1 mM). After 1 h of induction at room temperature, cells were collected by centrifugation, resuspended in lysis buffer (150 mM NaCl, 1 mM dithiothreitol, 50 mM EDTA, 2% Sarkosyl, and 50 mM Tris, pH 7.5), and sonicated 15 min at 4 °C. Bacterial debris was removed by centrifugation. 4% Triton X-100, 1 mM PEFABLOC (Interchim) and 2.5 mM benzamidine were added to the supernatant and applied to a glutathione S-transferase affinity column. After washes, the purified protein was eluted by thrombin digestion, as described by the manufacturer (Calbiochem) and then dialyzed against 50 mM Tris, pH 7.5, 150 mM NaCl, and 5 mM phenylmethylsulfonyl fluoride. Purified SRY protein was checked by SDS-PAGE analysis and immunoblotting, aliquoted with 2 mg/ml bovine serum albumin, and 50% glycerol and then stored at -80 °C. Mutant forms of SRY gene were produced using the Transformer Mutagenesis kit (CLONTECH, Ozyme, France).

In Vitro Phosphorylation and Immunoprecipitation-- 250 ng of bacterially expressed SRY protein were added to a standard PKA reaction mixture containing 20 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM unlabeled ATP, 5 mM dithiothreitol, 100 mM NaCl, and 1 mM [gamma -32P]ATP (0.5 Ci/mmol) and incubated either with 1 milliunit of PKA catalytic subunit (Promega, 1 unit = 1 mmol of phosphate/min) or with 2.5 µg of NT2/D1 nuclear extract in a final volume of 20 µl at 30 °C for 30 min. In the latter case, nuclear extract was prepared according to Dignam et al. (29) and SRY protein was immunoprecipitated with SRY Y127.3 antibody as described previously (17). Reactions were stopped by the addition of 20 µl of 1× Laemmli sample buffer, and protein was separated by gel electrophoresis and submitted to autoradiography.

DNA-binding Assays-- Gel mobility shift assays were carried out with 250 ng of wild-type or mutant SRY proteins after phosphorylation with 25 units of PKA catalytic subunit or after incubation with 2.5 µg of NT2/D1 nuclear extract with 10,000 cpm of either a double-stranded SRY DNA-binding consensus site termed SRBS (5'-GATCTATCCCGAACAATTTCACAGCT) or an unrelated DNA sequence termed unr (5'-AGCTGCACCGCCCCCGCCAGGAGG) labeled by a fill-in reaction with (alpha -32P) dCTP and purified by non-denaturating polyacrylamide electrophoresis. For a typical binding reaction, incubation was performed in a volume of 20 µl containing 20 mM HEPES, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 400 ng of poly(dI-dC) for 30 min at room temperature in the presence or not of the corresponding antibody. The samples were then electrophoresed through a non-denaturing 6% polyacrylamide gel run in 0.5 × TBE at 4 °C and submitted to phosphoimager analysis after fixing.

Kinetic Analysis of SRY DNA-binding after PKA Phosphorylation-- The kinase reactions were performed as described above except that no radiolabeled ATP was added. The reactions were stopped after 0.5, 1, 2, 5, and 20 min by the addition of 1 µg of PKA kinase inhibitor (Sigma) and transferred on ice. The phosphorylated proteins were subjected to a gel mobility shift assay with the same oligonucleotide described in the previous paragraph. As a control of the protein kinase A inhibitor (PKI) activity, the same reactions were carried out in the presence of [gamma -32P]ATP, stopped as above, and run on SDS-PAGE electrophoresis. DNA-binding activity and phosphate incorporation were quantified by phosphoimager analysis.

Immunofluorescence Studies-- Cells were fixed with methanol at -20 °C for five minutes and rehydrated with phosphate-buffered saline (PBS) at room temperature. All incubations were performed in 1% PBS-bovine serum albumin, and washings were in PBS. After a 30-min preincubation at room temperature, cells were immunostained for 1 h in a humid chamber at 37 °C with the rabbit SRY antibody (diluted 1/100) as described previously (12). Cells were washed in PBS, and primary antibodies were visualized with biotinylated anti-rabbit (dilution 1/200) and Texas red-conjugated streptavidin antibodies (dilution 1/200) (Amersham). In each case, incubations were performed in the same conditions described for the primary antibody. Nuclei were visualized using Hoechst 33286 DNA staining (30). Cells were washed again and mounted in FluorSave reagent (Calbiochem). Images were collected and processed on a Bio-Rad confocal microscope or on a Zeiss Axiophot.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The SRY Protein Is Phosphorylated in Vivo on Serine Residue(s)-- We first examined the phosphorylation state of SRY in human NT2/D1 cells. This cell line was chosen because we, and others, have previously described that these cells scored positive for SRY expression (12, 31). NT2/D1 cells were labeled with [32P]orthophosphate, and after cell disruption, SRY was immunoprecipitated with Y127.3 antibody, an antibody raised against an SRY-derived peptide with a previously demonstrated specificity (12). A polypeptide with Mr = 27,000, corresponding to the expected size of SRY, was immunoprecipitated from cell extracts as visualized by SDS-PAGE and autoradiography (Fig. 1A). This signal was specific since no signal was observed when using preimmune antisera or when the anti-SRY was saturated with bacterial extracts synthesizing SRY (data not shown). We therefore conclude that SRY is a phosphorylated polypeptide in NT2/D1 cells. To identify the phosphorylated residues, the immunoprecipitated SRY protein was eluted from the SDS-PAGE and subjected to phosphoamino acid analysis. An autoradiogram of the thin layer chromatography plate showed only phosphoserine from the SRY protein, even after very long exposure (Fig. 1B). This result reveals that the primary in vivo phosphorylation site of human SRY is restricted to serine residue(s).


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Fig. 1.   Human SRY is phosphorylated in vivo on serine. A, phosphorylation of SRY in vivo. NT2/D1 cells were labeled with 32P inorganic phosphate for 30 min, and SRY was immunoprecipitated with anti-SRY resolved by SDS-PAGE and autoradiography (2nd lane). The specificity of the precipitation was assessed by the use of pre-immune serum (1st lane). The sizes of marker proteins, in kilodaltons, are indicated. B, phosphoamino acid analysis of phosphorylated SRY. Shown is an autoradiogram of the thin layer chromatography plate on which the phosphoamino acids were separated. The positions of unlabeled phosphoamino acid standards detected by ninhydrin are indicated (Ser (P), phosphoserine; Thr (P), phosphothreonine; Tyr (P), phosphotyrosine).

Consensus Phosphorylation Sites Are Present in the Human SRY Protein Sequence-- Computer analysis of the human SRY protein sequence for consensus motifs of phosphorylation by different kinases reveals three putative sites: 1) a PKA phosphorylation site in the N-terminal part (29RRSSS33) (32, 33), 2) a casein kinase II (CKII) site located in the HMG domain involving a threonine as a potential phosphoamino acid (102TEAE105) (33, 34), and 3) a weak consensus CKII site in the C-terminal part (174SRME177) of the SRY protein (Fig. 2A). While the first two sites are both perfectly consensual, our previous phosphoamino acid analysis argues for a possible physiological role of the PKA phosphorylation site in the SRY sequence, where three serine residues are present.


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Fig. 2.   Phosphorylation of human SRY by PKA in vitro. A, amino acid sequence of the human SRY protein. The HMG domain of the protein is highlighted as bold lettering. The potential PKA phosphorylation site is boxed, and the two CKII sites are underlined; all three were established by computer analysis using the computer program PROSITE dictionary (Dr. Amos Bairoch, University of Geneva, Switzerland). The three serine residues substituted by alanines in the mutant SRY (SRY-Ala) are indicated by an asterisk. B, bacterially expressed and purified human SRY was used in a kinase assay with PKA catalytic subunit, with (lane 2) or without (lane 1) the PKA inhibitor PKI. PKA catalytic subunit alone was used as control (lane 4). The reactions were then subjected to SDS-PAGE with subsequent autoradiography.

SRY Serves As an in Vitro Substrate for PKA-- The presence of this PKA phosphorylation site in the SRY sequence suggests that this kinase may directly phosphorylate the SRY protein. To test this hypothesis, full-length SRY protein was expressed in bacteria and purified (see "Materials and Methods"). The purified protein was then tested in an in vitro kinase assay with the PKA catalytic subunit. As shown in Fig. 2B, SRY protein served as a substrate for PKA, and this phosphorylation was abolished in the presence of PKI.

SRY Serves As a Substrate for PKA Activity from NT2/D1 Cell Nuclear Extracts-- Taken together, the results described above strongly indicate that the human SRY protein might be phosphorylated by PKA in vivo. To substantiate the relevance of this phosphorylation event, phosphorylation of exogenous human SRY protein was next investigated using NT2/D1 cell nuclear extracts. Endogenous SRY protein was absent from these extracts since the classical method used to prepare nuclear extracts (see "Materials and Methods") did not permit its separation from chromatin, as tested using Western blotting analysis (data not shown). It was thus possible to specifically follow the phosphorylation of exogenous SRY protein. After incubation in the nuclear extracts in the presence of (gamma -32P)ATP, the bacterially expressed SRY protein was immunoprecipitated with Y127.3 antibody, and its phosphorylation state was estimated by SDS-PAGE and autoradiography (Fig. 3A). SRY was phosphorylated in NT2/D1 nuclear extract, and this phosphorylation was efficiently inhibited by PKI. In the same way, an excess of PKA regulatory subunits (R-sub) in the absence of cAMP abolished SRY phosphorylation, but in this case, phosphorylation was restored in the presence of cAMP (Fig. 3A).


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Fig. 3.   SRY phosphorylation in NT2/D1 nuclear extract is PKA-dependent. A, immunoprecipitation of SRY (wild-type or mutant) after incubation in [gamma -32P]ATP containing NT2/D1 nuclear extract as described under "Materials and Methods." Shown are phosphorylation of the wild-type SRY protein (SRY WT) alone (lane 1), or in the presence of 500 ng of PKA inhibitor (PKI) (lane 2), or in the presence of 100 units of PKA regulatory subunit (R-sub, Promega) plus 10 µm cAMP (lane 3) or with 100 units of PKA regulatory subunit only. Lane 5, control in absence of SRY; lanes 6 and 7, similar experiments using the mutant SRY protein (SRY-Ala). B, stimulation of SRY in 8-Br-cAMP-treated intact NT2/D1 cells. Shown is the immunoprecipitation of the SRY protein without (lane 1) or after 1 mM 8-Br-cAMP treatment (lane 2). Pre-immune serum (lanes 3 and 4) was used as control.

For further identification of the PKA phosphorylation site, we performed tryptic or V8 phosphopeptide mapping studies with labeled SRY protein, but unfortunately, these experiments led only to partial digests of the immunoprecipitated SRY. To allow the identification of the phosphorylation site in the intact native SRY protein, we then carried out site-directed mutagenesis of the three serine residues from the PKA phosphorylation site (Fig. 2A) to alanine. The mutant protein (SRY-Ala) was expressed in bacteria, purified, and utilized for in vitro phosphorylation reactions using NT2/D1 nuclear extracts (Fig. 3A). Unlike the native SRY protein, triple mutated SRY was not a substrate of PKA (Fig. 3A) as previously observed when using purified PKA (data not shown). This result confirms that one (or more) of the three mutated serine residues of the N-terminal domain of SRY was the site for PKA phosphorylation.

Stimulation of SRY Phosphorylation in 8-Bromo-cAMP-treated Intact Cells-- To investigate whether activation of the PKA pathway in intact cells alters SRY phosphorylation, [32P]orthophosphate-labeled NT2/D1 cells were stimulated with 8-bromo-cAMP (8-Br-cAMP), a strong activator of PKA, in the presence of 1 mM isobutylmethylxanthine (IBMX). After stimulation, the cells were lysed, and the newly labeled SRY protein was immunoprecipitated. As expected, in the presence of 8-Br-cAMP, immunoprecipitated SRY protein was phosphorylated to a greater extent than in non-stimulated cells (Fig. 3B). The phosphorylated state of SRY observed in non-stimulated cells could represent the basal level of SRY phosphorylation by PKA and/or other kinases.

In summary, the difference of phosphorylation between 8-Br-cAMP-stimulated and control cells clearly demonstrated that SRY protein is the subject of PKA phosphorylation.

PKA Phosphorylation Enhances the DNA-binding Activity of SRY-- To test the hypothesis that PKA-phosphorylated SRY protein exhibits altered DNA-binding activities, SRY was phosphorylated in the presence of NT2/D1 cell nuclear extracts mixed with the appropriate SRY oligonucleotide target and then subjected to gel shift assay (Fig. 4A, lane 2). Y127.3 antibody caused the complex to migrate more slowly in the gel-shift assay, demonstrating further that the DNA-binding of the NT2/D1 nuclear extract is indeed due to the SRY protein (Fig. 4A, lane 3). A clear reduction in DNA binding was detected when SRY was preincubated with the same nuclear extract plus PKI (Fig. 4A, lane 1). An approximately 10-fold reduction was observed in the presence of PKI as estimated using phosphoimager quantification. Similar results were observed when SRY protein was incubated with PKA in the absence of nuclear extracts (Fig. 4A, lanes 5, 6, and 7). To assess the specificity of the SRY·DNA complex formation in NT2/D1 extracts, homologous unlabeled probe was shown to compete efficiently with complex formation (Fig. 4A, lane 9 versus lane 8). By contrast, a nonsimilar oligonucleotide failed to displace the complex (Fig. 4A, lane 10). In the same way, an irrelevant antibody failed to supershift the complex (Fig. 4A, lane 12 versus lane 11). Finally, when no purified SRY protein was added to NT2/D1 cell extracts, no complex was observed.


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Fig. 4.   Gel mobility shift assays using phosphorylated and nonphosphorylated SRY proteins. Bacterially expressed and purified wild-type (A) or mutant SRY proteins (B) were incubated in the presence of NT2/D1 cell nuclear protein extracts or with purified PKA catalytic subunit. These proteins were then incubated with a 32P-labeled oligonucleotide including an SRY binding site (SRBS) as a probe, either alone, or in the presence of cold SRBS oligonucleotide or of unrelated oligonucleotide (unr) used in each case as competitor. Electrophoresis was then performed through a 6% nondenaturing polyacrylamide gel. Lane 4 in panels A and B contains probe only. Abbreviations used are: SRY WT, SRY wild-type protein; NT2 ext, NT2/D1-cell nuclear extract; alpha SRYab, SRY antibody; unr ab, unrelated purified antibody; PKI, protein kinase A inhibitor; SRY-Ala, triple mutation Ser right-arrow Ala; SRBS, SRY DNA-binding site containing oligonucleotide; unr, unrelated DNA-binding site containing oligonucleotide; comp, competition with. Arrows correspond to SRY-DNA complex, and asterisks correspond to the supershift complex.

To confirm that the PKA phosphorylation site present in the N-terminal part of SRY is required to regulate the DNA-binding activity of the SRY protein, we performed the same experiments with the mutant SRY protein described above (SRY-Ala). As shown in Fig. 4B (lanes 1-6), neither NT2/D1 nuclear extracts nor purified PKA were able to enhance the DNA-binding activity of the mutant SRY demonstrating that PKA phosphorylation of the N-terminal part is required to enhance the DNA-binding activity of SRY. To assess the specificity of the complex observed, the same control experiments as in Fig. 4A (lanes 8-13) were performed (Fig. 4B, lanes 7-12). Finally, when compared, the kinetics of DNA-binding of SRY in the presence of PKA and the kinetics of SRY phosphorylation are clearly correlated (Fig. 5).


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Fig. 5.   Comparison of the time course of SRY phosphorylation by PKA with the time course of its resulting effect on DNA-binding activity. A, bacterially expressed SRY was subjected to kinase reactions for varying times (see "Materials and Methods"), and the reactions were stopped with 1 µg of PKA inhibitor (PKI), followed by SDS-PAGE and analyzed with a phosphoimager. Control (lane 1) was obtained in the absence of PKA for 20 min. B, kinetics of SRY DNA-binding activity after PKA phosphorylation. Phosphorylations were performed as previously (see panel A) in the absence of labeled ATP, and the reactions were next subjected to gel mobility shift assays as described under "Materials and Methods." Control (lane 1) was obtained in the absence of PKA.

From these experiments, we can conclude that the PKA phosphorylation directly activates the DNA-binding activity of the SRY protein via a phosphorylation site located in its N-terminal domain, providing, for the first time, a regulatory role to this domain of the SRY protein.

Modulation of Transcription by SRY Is Stimulated by PKA-- In an effort to establish the effectiveness of the PKA phosphorylation, we also examined whether this phosphorylation event may play a role in regulating SRY transcriptional activity. While no target gene has been assigned to SRY so far, the ability of SRY to structurally reorganize regions of the chromatin, via its DNA-binding, leading to repression of the stimulatory activity of CREB on the c-fos promoter has been described (35). For these studies and to simplify the assays, COS7 cells were cotransfected with multimerized SRY binding sites upstream of a minimum thymidine kinase promoter-CAT reporter (pTK(56)7; Ref. 36) along with an SRY expression vector (pJ3Omega -SRY). In this case, basal thymidine kinase promoter activity was inhibited approximately 50% in cells overexpressing SRY (Fig. 6). Transrepressor activity was clearly dependent upon the DNA-binding activity of SRY since a mutant SRY protein (SRY*), previously described to be unable to bind DNA (37), failed to inhibit CAT activity. Similarly cotransfection of the SRY expression vector with the reporter plasmid construct lacking copies of the DNA-binding site p(Control) failed to affect CAT activity (Fig. 6). It was then pertinent to know whether this repression activity of SRY and then the DNA binding activity of SRY in vivo was affected by its phosphorylation status. The results of the transient transfection CAT assays after 8-Br-cAMP stimulation of the COS7 cells showed a repression to an extent greater than that detected without any stimulation (approximately 75%) (Fig. 6). The SRY-Ala mutant protein slightly affected basal promoter activity (only 10%), and as expected, these Ser right-arrow Ala substitutions within the PKA phosphorylation site of SRY abolished stimulation of repression when compared with the SRY wild-type. As proven using immunofluorescence analysis, the inability of this mutant SRY-Ala to repress transcription is not the result of an alteration in its subcellular localization since it showed a similar nuclear staining to the wild-type (Fig. 7).


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Fig. 6.   SRY acts as a repressor on CAT plasmids under the control of multimerized SRY binding sites upstream of a minimum thymidine kinase promoter (pTK(56)7). COS7 cells were transfected with the indicated plasmids using the LipofectAMINE method as described under "Materials and Methods." CAT activity is reported as percent of activity relative to basal promoter activity obtained in the SRY protein with the empty pJ3Omega expression plasmid transfected in combination with the pTK(56)7 reporter plasmid, or with the same reporter plasmid devoid of SRY binding sites p(Control). When indicated, COS7 cells were treated with 1 mM 8-Br-cAMP. The error bars represent the standard deviation of three independent transfections of the indicated sample. SRY, wild-type SRY protein; SRY*, mutated SRY protein (Y127 right-arrow C127) without any DNA-binding capacity; SRY-Ala, mutated SRY protein with the triple mutation Ser right-arrow Ala. When indicated, values were significantly different (xxx, p <=  0.005) as determined by Student's t-test.


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Fig. 7.   The wild-type SRY protein (SRY WT, panel a) and the mutant SRY (SRY-Ala, panel c) are located in the cell nucleus. Plasmids encoding the full-length SRY open reading frame (pJ3Omega -SRY) or a mutant unable to be phosphorylated by PKA were transfected in COS7 cells. After fixing, the subcellular localization of the SRY proteins was determined by indirect immunofluorescence with the anti-SRY antibody Y127.3. Panels b and d correspond to the characterization of the nuclei of the same cells using Hoechst 33286 to visualize DNA.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein modification provides a direct means of regulating the activities of transcription factors in response to specific signals. A number of post-translational modifications may be involved, but phosphorylation is recognized as one of the principal means by which the activities of transcription factors can be regulated (19). Phosphorylation can act at multiple levels, including DNA binding activity, transcriptional regulation, nuclear transport, or dimerization. In this study, we have shown that human SRY, which plays a critical role in mammalian male sex determination, is the target of such a phosphorylation event. The human SRY protein was shown to be 204 amino acids long with a central DNA binding domain belonging to the high mobility group of proteins (2). Upon binding, SRY induces a strong bending of the target DNA and is thus likely to have a structural role by locally altering the chromatin structure (13). In this study, we first analyzed the phosphorylation state of SRY in NT2/D1 cells by immunoprecipitation experiments using specific SRY antibody that has been previously characterized (12). This human NT2/D1 cell line has been shown by different authors to endogenously express SRY (12, 31) but also the genes described so far, as implicated in the sex determining pathway.2 Then this cell line constitutes a good cell model to study SRY regulation in human. We thus showed that, in this cell line, human SRY is indeed a phosphoprotein. By phosphoamino acid analysis, we also demonstrated that this phosphorylation occurred solely on serine residue(s) in vivo. Analysis of human SRY primary amino acid sequence confirmed the existence of a consensus site for protein kinase A lying just upstream of the SRY DNA-binding domain. The purified, bacterially expressed protein was indeed phosphorylated by protein kinase A or by nuclear extracts from NT2/D1 cells in a PKI-sensitive manner. Mutational analysis confirmed the importance of one, two, or maybe three serine residues close together within the N-terminal part of the human SRY protein, next to the DNA binding domain. We have demonstrated that this phosphorylation event thus can be enhanced upon activation of the cAMP signaling pathway using 8-Bromo-cAMP as a stimulating agent, in the NT2/D1 carcinoma cell line, demonstrating the physiological relevance of this phosphorylation. Phosphorylation can act positively or negatively on the binding of sequence-specific transcription factors to DNA (19). For the human SRY, we were able to show that the DNA-binding activity of the protein produced in bacteria can be greatly enhanced, as estimated by gel shift analysis, after phosphorylation with the PKA catalytic subunit or with nuclear extracts. These findings are in good agreement with previous results reported (38) where the addition of Chinese hamster ovary (CHO)-cell nuclear extracts was shown to enhance SRY binding activity, suggesting the existence of a factor present in the extracts with the ability to enhance or to stabilize DNA binding by SRY. Until now, the number of transcription factors whose DNA-binding activity is stimulated after phosphorylation remains low and serum response factor (SRF), via phosphorylation of serine residues in its N-terminal part close to the DNA-binding domain, is one of the rare examples (39). SRY constitutes now a new example of this transcription factor subclass. Further enzymatic studies will be necessary to understand if the PKA phosphorylation stabilizes the protein-DNA complex, affects the association kinetics, or both together.

Finally, our results from the in vivo transfection assays in COS cells are consistent with the fact that PKA phosphorylation is probably crucial for SRY DNA-binding activity in vivo, as demonstrated using the mutant form of the protein, SRY-Ala. Indeed, while the mutant SRY still binds DNA in vitro, its in vivo activity, as measured by our transfection assay, is rather low. These results are also consistent with the conclusion that the human SRY protein is able to act as a repressor in assays involving concatamerized DNA target sequences for SRY-promoter-CAT constructs. Though the repression activity described here fits well with the recent hypothesis of McElreavey et al. (40), its physiological relevance will require confirmation in vivo, on a SRY target gene, as soon as one is identified. Furthermore, the repressor activity of SRY was enhanced upon activation of the PKA transduction pathway, in parallel with its DNA binding activity. The importance of the DNA binding activity of the sequence-specific transcription factor SRY via its HMG domain is well described (41), but recent data have also shown that the binding affinity of an HMG box can be substantially increased by the presence of amino acid terminal sequences on both sides of the box (42). Our results suggest a rather simple model according to which phosphorylation of SRY N-terminal sequence leads to an increase in the affinity of the protein for the cognate target DNA sequences, which will then mediate SRY biological function. At this step, further studies are now required to understand whether phosphorylation enhances SRY binding activity by altering the conformation of its HMG domain or by providing additional contacts that could be critical for the protein-DNA interaction.

Despite a rapid divergence between mammalian Sry sequences (43), sequence comparison reveals that the PKA site identified in this study is perfectly conserved across primates and remains optimal. Furthermore in all eutherian mammals, except rodents, phosphorylatable residues (serine or threonine) remain conserved in the N-terminal part of the molecule though their phosphorylation status still needs to be analyzed.

In conclusion, this study raises the possibility that a cAMP-dependent signal pathway controls the human testis determining factor activity. Nevertheless we cannot exclude additional phosphorylation(s) by other kinase activities or phosphorylation events too transient to be detected in our assays. We show here that this mode of control affects the SRY protein activity itself and may represent a crucial event in the chromatin reorganization occurring as SRY binds DNA. The existence of a putative cAMP response element upstream of the SRY open reading frame (44) has also pointed to the possible control of this cascade via this cAMP-dependent transduction pathway, but in this instance at the level of SRY gene expression itself. Further studies are now required to unravel which signal transduction mechanisms control SRY activity. This will provide essential clues to how sex determination is regulated during early development.

Acknowledgments-- We thank Dr. Daniel Fisher for critical reading of the manuscript and Dr. Hans Clevers for providing us with the pTK(56)7 reporter plasmid construct. P. T. thanks A. Fernandez and N. Lamb for support.

    FOOTNOTES

* This work was supported by CNRS and in part by the BIOMED 2 program from the European Economic Community.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.

Dagger Contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 33 4 67 61 33 49; Fax: 33 4 67 52 15 59; E-mail: berta{at}vega.crbm.cnrs-mop.fr.

1 The abbreviations used are: TDF, testis determining factor; PKA, protein kinase A; HMG, high mobility group; PAGE, polyacrylamide gel electrophoresis; unr, unrelated; TBE, Tris borate-EDTA; PKI, protein kinase A inhibitor; PBS, phosphate-buffered saline; CKII, casein kinase II; R-sub, regulatory subunits; 8-Br-cAMP, 8-bromo-cAMP.

2 Marion Desclozeaux, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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