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

Abstract

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. 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. 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 (1Greenfield A. Koopman P. Curr. Top. Dev. Biol. 1996; 34: 1-23Crossref PubMed Scopus (13) Google Scholar). In 1990, the SRY (sex determining region of Y) gene was isolated from the human Y chromosome (2Sinclair A.H. Berta P. Palmer M.S. Hawkins J.R. Griffiths B.L. Smith M.J. Foster J.W. Frischauf A.M. Lovell-Badge R. Goodfellow P.N. Nature. 1990; 346: 240-244Crossref PubMed Scopus (2684) Google Scholar) and was subsequently shown to equate with the elusive testis determining factor (TDF) 1The 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. (3Berta P. Hawkins J.R. Sinclair A.H. Taylor A. Griffiths B.L. Goodfellow P.N. Fellous M. Nature. 1990; 348: 448-450Crossref PubMed Scopus (428) Google Scholar, 4Koopman P. Gubbay J. Vivian N. Goodfellow P.N. Lovell-Badge R. Nature. 1991; 351: 117-121Crossref PubMed Scopus (1794) Google Scholar). 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 (5Laudet V. Stehelin D. Clevers H. Nucleic Acids Res. 1993; 21: 2493-2501Crossref PubMed Scopus (291) Google Scholar). 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 (6van de Wetering M. Oosterwegel M. Dooijes D. Clevers H. EMBO J. 1991; 10: 123-132Crossref PubMed Scopus (464) Google Scholar) and LEF-1 (7Travis A. Amsterdam A. Belanger C. Grosschedl R. Genes Dev. 1991; 5: 880-894Crossref PubMed Scopus (504) Google Scholar, 8Giese K. Amsterdam A. Grosschedl R. Genes Dev. 1991; 5: 2567-2578Crossref PubMed Scopus (227) Google Scholar), or diverse SOX (SRY-box related) proteins (9Pevny L.H. Lovell-Badge R. Curr. Opin. Genet. Dev. 1997; 7: 338-344Crossref PubMed Scopus (420) Google Scholar). In vitro binding assays show that the SRY HMG box exhibits sequence-specific DNA-binding to the consensus sequence AACAAT (10Harley V.R. Lovell-Badge R. Goodfellow P.N. Nucleic Acids Res. 1994; 22: 1500-1501Crossref PubMed Scopus (336) Google Scholar) involving interaction in the DNA minor groove (11van de Wetering M. Clevers H. EMBO J. 1992; 11: 3039-3044Crossref PubMed Scopus (177) Google Scholar). In addition, the SRY protein was shown to be nuclear (12Poulat F. Girard F. Goze C. Rebillard X. Heitz F. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar) and to induce a strong bending of the DNA target (13Ferrari S. Harley V.R. Pontiggia A. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1992; 11: 4497-4506Crossref PubMed Scopus (383) Google Scholar). 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 (9Pevny L.H. Lovell-Badge R. Curr. Opin. Genet. Dev. 1997; 7: 338-344Crossref PubMed Scopus (420) Google Scholar), 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 (14Koopman P. Reprod. Fertil. Dev. 1995; 7: 713-722Crossref PubMed Scopus (31) Google Scholar). 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 (15Yuan H. Corbi N. Basilico C. Dailey L. Genes Dev. 1995; 9: 2635-2645Crossref PubMed Scopus (646) Google Scholar, 16Giese K. Grosschedl R. EMBO J. 1993; 12: 4667-4676Crossref PubMed Scopus (140) Google Scholar), 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 (17Poulat F. de Santa Barbara P. Desclozeaux M. Soullier S. Moniot B. Bonneaud N. Boizet B. Berta P. J. Biol. Chem. 1997; 272: 7167-7172Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), or the demonstration of potential SRY-calmodulin binding (18Harley V.R. Lovell-Badge R. Goodfellow P.N. Hextall P.J. FEBS Lett. 1996; 391: 24-28Crossref PubMed Scopus (69) Google Scholar). 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 (20Ye Y. Raychaudhuri B. Gurney A. Campbell C.E. Williams B.R.G. EMBO J. 1996; 15: 5606-5615Crossref PubMed Scopus (86) Google Scholar, 21Moll U.M. Riou G. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7262-7266Crossref PubMed Scopus (539) Google Scholar), its transactivation potential (22Smeal T. Hibi M. Karin M. EMBO J. 1994; 13: 6006-6010Crossref PubMed Scopus (86) Google Scholar, 23Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 5: 544-547Crossref Scopus (303) Google Scholar), its DNA-binding activity (24Marais R.M. Hsuan J.J. McGuigan C. Wynne J. Treisman R. EMBO J. 1992; 11: 97-105Crossref PubMed Scopus (128) Google Scholar, 25Lüscher B. Christenson E. Litchfield D.W. Krebs E.G. Eisenman R.N. Nature. 1990; 344: 517-522Crossref PubMed Scopus (310) Google Scholar), 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. 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 mmglutamine 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) pJ3Ω expression plasmid (26Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 1068Crossref PubMed Scopus (278) Google Scholar) 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 using by as PubMed Scopus Google Scholar). SRY was by and to The to SRY was from the in 6 for at and to acid analysis as T. L.H. P. J.R. Karin M. Hunter T. 1991; Full Text PDF PubMed Scopus Google Scholar). The was with 1 μg of and as The was next a and by high acid were with and with the by of the of SRY protein using diverse expression the of the human SRY were replaced by DNA, to with The SRY was then in the expression The protein was in the after by After 1 h of at cells were by in 1 and and at was by 1 and were to the and to a After the purified protein was by as by the and then and SRY protein was by analysis and with 2 serum and and then at of SRY gene were using the France). ng of SRY protein were to a PKA containing 1 and 1 and either with 1 of PKA 1 1 of or with μg of NT2/D1 nuclear in a of μl at °C for In the nuclear was to PubMed Scopus Google Scholar) and SRY protein was with SRY as (17Poulat F. de Santa Barbara P. Desclozeaux M. Soullier S. Moniot B. Bonneaud N. Boizet B. Berta P. J. Biol. Chem. 1997; 272: 7167-7172Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). were by the addition of μl of and protein was by gel and to mobility shift assays were with ng of or mutant SRY proteins after phosphorylation with of PKA or after with μg of NT2/D1 nuclear with of either a SRY DNA-binding consensus site or an DNA sequence by a with and purified by polyacrylamide a binding was performed in a of μl containing and ng of for at in the presence or of the The were then a polyacrylamide gel in at °C and to analysis after The kinase were performed as that no was The were after 2, and by the addition of 1 μg of PKA kinase and on The phosphorylated proteins were to a gel mobility shift with the in the a control of the protein kinase A activity, the were in the presence of as and on DNA-binding activity and were by were with at °C for and with phosphate-buffered at All were performed in serum and were in After a at cells were for 1 h in a at 37 °C with the SRY as (12Poulat F. Girard F. Goze C. Rebillard X. Heitz F. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar). were washed in PBS, and were with and In were performed in the for the were using DNA M. EMBO J. PubMed Scopus Google Scholar). were washed and in reagent were and on a or on a We the phosphorylation of SRY in human NT2/D1 cells. This cell was and have that these cells for SRY expression (12Poulat F. Girard F. Goze C. Rebillard X. Heitz F. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar, C. A.J. Sinclair A.H. Palmer M.S. Lovell-Badge R. Goodfellow P.N. Genet. 1993; PubMed Scopus Google Scholar). NT2/D1 cells were with and after cell SRY was with an an with a demonstrated specificity (12Poulat F. Girard F. Goze C. Rebillard X. Heitz F. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar). A with to the of SRY, was from cell as by and This was no was when using or when the was with SRY We that SRY is a phosphorylated in NT2/D1 cells. To the phosphorylated the SRY protein was from the and to acid of the from the SRY after 1 This result that the in vivo phosphorylation site of human SRY is to serine analysis of the human SRY protein sequence for consensus of phosphorylation by a PKA phosphorylation site in the N-terminal part J.R. Krebs E.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 1991; PubMed Scopus Google Scholar), a casein kinase site located in the HMG domain involving a as a potential acid 1991; PubMed Scopus Google Scholar, 1990; PubMed Scopus Google Scholar), and a consensus site in the C-terminal part of the SRY protein 2 the are acid analysis for a role of the PKA phosphorylation site in the SRY serine residues are The presence of this PKA phosphorylation site in the SRY sequence that this kinase the SRY protein. To this SRY protein was in and purified and The purified protein was then in an in vitro kinase with the PKA shown in 2 SRY protein as a substrate for PKA, and this phosphorylation was in the presence of the results strongly that the human SRY protein be phosphorylated by To the of this phosphorylation phosphorylation of human SRY protein was next investigated using NT2/D1 cell nuclear SRY protein was from these the used to nuclear and its from as using analysis was to specifically the phosphorylation of SRY protein. After in the nuclear in the presence of the SRY protein was with and its phosphorylation was by and SRY was phosphorylated in NT2/D1 nuclear and this phosphorylation was by In the an of PKA regulatory in the of SRY but in this phosphorylation was in the presence of further of the PKA phosphorylation we performed or studies with SRY but these led to of the To the of the phosphorylation site in the SRY we then of the serine residues from the PKA phosphorylation site 2 to The mutant protein was in and for in vitro phosphorylation using NT2/D1 nuclear the SRY SRY was a substrate of PKA as when using purified PKA This result that one of the serine residues of the N-terminal domain of SRY was the site for PKA phosphorylation. To whether of the PKA pathway in cells SRY NT2/D1 cells were with a strong of PKA, in the presence of 1 After the cells were and the SRY protein was in the presence of 8-Br-cAMP, SRY protein was phosphorylated to a in cells The phosphorylated of SRY in cells could the basal of SRY phosphorylation by PKA other In the of phosphorylation and control cells demonstrated that SRY protein is the of PKA phosphorylation. To the hypothesis that SRY protein exhibits DNA-binding SRY was phosphorylated in the presence of NT2/D1 cell nuclear with the SRY target and then to gel shift the to in the assay, further that the DNA-binding of the NT2/D1 nuclear is to the SRY protein A in DNA binding was when SRY was with the nuclear was in the presence of as using results were when SRY protein was with PKA in the of nuclear and To the specificity of the in NT2/D1 was shown to with contrast, a failed to the In the an failed to the when no purified SRY protein was to NT2/D1 cell no was To that the PKA phosphorylation site present in the N-terminal part of SRY is to regulate the DNA-binding activity of the SRY we performed the with the mutant SRY protein shown in NT2/D1 nuclear purified PKA were to enhance the DNA-binding activity of the mutant SRY that PKA phosphorylation of the N-terminal part is to enhance the DNA-binding activity of To the specificity of the the control as in were performed when the of DNA-binding of SRY in the presence of PKA and the of SRY phosphorylation are these we can that the PKA phosphorylation the DNA-binding activity of the SRY protein via a phosphorylation site located in its N-terminal for the a regulatory role to this domain of the SRY protein. In an to the of the PKA we also whether this phosphorylation event play a role in SRY transcriptional no target gene has been to SRY the ability of SRY to of the via its DNA-binding, to of the activity of on the promoter has been S. Genes Dev. 1993; 7: PubMed Scopus Google Scholar). these studies and to the COS7 cells were with SRY binding upstream of a thymidine kinase reporter de Wetering M. Oosterwegel M. van K. Clevers H. EMBO J. 1993; 12: PubMed Scopus Google Scholar) with an SRY expression In this basal thymidine kinase promoter activity was in cells SRY activity was the DNA-binding activity of SRY a mutant SRY protein to be to bind DNA F. Soullier S. Goze C. Heitz F. Calas B. Berta P. 1994; PubMed Scopus Google Scholar), failed to inhibit CAT of the SRY expression with the reporter plasmid of the DNA-binding site failed to affect CAT activity was then to whether this activity of SRY and then the DNA binding activity of vivo was affected by its phosphorylation The results of the transfection CAT assays after of the COS7 cells a to an that The mutant protein affected basal promoter activity and as these the PKA phosphorylation site of SRY of when with the SRY using the of this mutant to transcription is the result of an in its subcellular localization it a nuclear to the SRY protein and the mutant SRY are located in the cell Plasmids the SRY or a mutant to be phosphorylated by PKA were in COS7 cells. After the subcellular localization of the SRY proteins was by with the and to the of the of the cells using to a direct of the activities of transcription in to A of post-translational modifications be but phosphorylation is as one of the by which the activities of transcription can be T. Karin M. 1992; Full Text PDF PubMed Scopus Google Scholar). Phosphorylation can at multiple DNA binding activity, transcriptional nuclear or In this study, we have shown that human SRY, which a role in determination, is the target of such a phosphorylation The human SRY protein was shown to be with a DNA binding domain to the high mobility of proteins (2Sinclair A.H. Berta P. Palmer M.S. Hawkins J.R. Griffiths B.L. Smith M.J. Foster J.W. Frischauf A.M. Lovell-Badge R. Goodfellow P.N. Nature. 1990; 346: 240-244Crossref PubMed Scopus (2684) Google Scholar). SRY induces a strong bending of the target DNA and is to have a role by altering the (13Ferrari S. Harley V.R. Pontiggia A. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1992; 11: 4497-4506Crossref PubMed Scopus (383) Google Scholar). In this study, we analyzed the phosphorylation of SRY in NT2/D1 cells by using SRY that has been (12Poulat F. Girard F. Goze C. Rebillard X. Heitz F. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar). This human NT2/D1 cell has been shown by to SRY (12Poulat F. Girard F. Goze C. Rebillard X. Heitz F. Calas B. Lamb N. Berta P. J. Cell Biol. 1995; 128: 737-748Crossref PubMed Scopus (101) Google Scholar, C. A.J. Sinclair A.H. Palmer M.S. Lovell-Badge R. Goodfellow P.N. Genet. 1993; PubMed Scopus Google Scholar) but also the as in the determining pathway. this cell a cell to SRY regulation in We in this cell line, human SRY is a acid we also demonstrated that this phosphorylation on serine in of human SRY acid sequence the of a consensus site for protein kinase A upstream of the SRY DNA-binding The protein was phosphorylated by protein kinase A or by nuclear from NT2/D1 cells in a analysis the of or serine residues the N-terminal part of the human SRY next to the DNA binding We have demonstrated that this phosphorylation event can be enhanced of the pathway using as a in the NT2/D1 carcinoma cell line, the of this phosphorylation. Phosphorylation can positively or on the binding of sequence-specific transcription to DNA T. Karin M. 1992; Full Text PDF PubMed Scopus Google Scholar). the human SRY, we were to show that the DNA-binding activity of the protein in can be as by gel shift after phosphorylation with the PKA or with nuclear are in with results reported Sinclair A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) the addition of nuclear was shown to enhance SRY binding activity, the of a factor present in the with the ability to enhance or to DNA binding by the of transcription DNA-binding activity is after phosphorylation and serum factor via phosphorylation of serine residues in its N-terminal part to the DNA-binding is one of the R. T. A. EMBO J. 1992; 11: PubMed Scopus Google Scholar). SRY a of this transcription factor studies will be to the PKA phosphorylation the protein-DNA the or results from the in vivo transfection assays in cells are consistent with the that PKA phosphorylation is probably for SRY DNA-binding activity in as demonstrated using the mutant of the the mutant SRY DNA in its in vivo activity, as by transfection assay, is results are also consistent with the that the human SRY protein is to as a repressor in assays involving DNA target for the activity well with the recent hypothesis of K. E. N. Fellous M. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar), its will in vivo on a SRY target as as one is the repressor activity of SRY was enhanced of the PKA in with its DNA binding The of the DNA binding activity of the sequence-specific transcription factor SRY via its HMG domain is well A. R. Harley V.R. Goodfellow P.N. Lovell-Badge R. Bianchi M.E. EMBO J. 1994; 13: PubMed Scopus Google Scholar), but recent have also shown that the binding of an HMG box can be by the presence of acid on of the box M. C. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar). results a to which phosphorylation of SRY N-terminal sequence to an increase in the of the protein for the target DNA which will then SRY biological function. this further studies are to whether phosphorylation enhances SRY binding activity by altering the of its HMG domain or by that could be for the protein-DNA a Lovell-Badge R. Goodfellow P.N. Nature. 1993; PubMed Scopus Google Scholar), sequence that the PKA site in this is and in all eutherian mammals, residues or in the N-terminal part of the their phosphorylation status to be In this the that a pathway the human testis determining factor we cannot by other kinase activities or phosphorylation to be in We show that this mode of control the SRY protein activity and a event in the as SRY The of a upstream of the SRY E. Fellous M. K. Cell Biol. 1992; Scholar) has also to the control of this via this but in this at the of SRY gene expression studies are to which mechanisms control SRY This will to determination is during We for of the and Clevers for with the reporter plasmid construct. P. T. A. and N. Lamb for