TFIIH Interacts with the Retinoic Acid Receptor γ and Phosphorylates Its AF-1-activating Domain through cdk7

Retinoic acid receptor γ (RARγ) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARγ1 and serines 66 and 68 in RARγ2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7·cyclin H·MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARγ is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARγ phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARγ transcriptional activity in a response gene-dependent manner. Retinoic acid receptor γ (RARγ) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARγ1 and serines 66 and 68 in RARγ2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7·cyclin H·MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARγ is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARγ phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARγ transcriptional activity in a response gene-dependent manner. retinoic acid receptor retinoid X receptor cdk-activating kinase human mouse polymerase chain reaction monoclonal antibody chloramphenicol acetyltransferase wild type polyacrylamide gel electrophoresis estrogen receptor mitogen-activated protein kinase The pleiotropic effects of retinoids are transduced by two nuclear receptor families, the retinoic acid receptors (RARs)1 and the retinoid X receptors (RXRs), that are ligand-dependent transregulators belonging to the nuclear receptor superfamily (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 2.Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (933) Google Scholar, 3.Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 4.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). RARs are activated by all-trans and 9-cis retinoic acid, whereas RXRs are activated by 9-cis retinoic acid only. There are three RAR (α, β, and γ) and three RXR (α, β, and γ) isotypes, and for each isotype there are at least two main isoforms that differ in their N-terminal region (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 5.Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 6.Chambon P. Semin. Cell Biol. 1994; 5: 115-125Crossref PubMed Scopus (499) Google Scholar).As do other members of the nuclear receptor superfamily, RARs and RXRs exhibit a conserved modular structure with six variably conserved regions (A to F) (Fig. 1) (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 5.Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). The N-terminal A/B region of RARs contains a ligand-independent transcriptional activation function, AF-1 (7.Nagpal S. Saunders M. Kastner P. Durand B. Nakshatri H. Chambon P. Cell. 1992; 70: 1007-1019Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 8.Nagpal S. Friant S. Nakshatri H. Chambon P. EMBO J. 1993; 12: 2349-2360Crossref PubMed Scopus (272) Google Scholar). Although the B regions of the three RAR isotypes are moderately conserved, their A regions are unrelated and differ for each isoform of a given RAR isotype (5.Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). The highly conserved C region encompasses the central DNA binding domain. The function of region F, if any, is unknown. Region E is more complex, as it contains the ligand binding domain, a dimerization interface, and the ligand-dependent transcriptional activation/repression domain AF-2 (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 9.Durand B. Saunders M. Gaudon C. Roy B. Losson R. Chambon P. EMBO J. 1994; 13: 5370-5382Crossref PubMed Scopus (315) Google Scholar). The activity of AF-2 is entirely dependent on the integrity of a conserved sequence referred to as the AF-2 AD core, located in α-helix 12 at the C-terminal end of the ligand binding domain. Ligand binding induces a major conformational change that includes helix 12 and creates a new surface for coactivator binding while corepressors are released, thus resulting in a transcriptional-competent nuclear receptor relayed to the transcriptional machinery and the chromatin template (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 10.Moras D. Gronemeyer H. Curr. Opin. Cell Biol. 1998; 10: 384-391Crossref PubMed Scopus (697) Google Scholar, 11.Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (509) Google Scholar, 12.Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (808) Google Scholar). The AF-2 and AF-1 activities synergize with each other in a response element- and promoter context-dependent manner (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 8.Nagpal S. Friant S. Nakshatri H. Chambon P. EMBO J. 1993; 12: 2349-2360Crossref PubMed Scopus (272) Google Scholar,13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Crossref PubMed Scopus (101) Google Scholar).RARs and RXRs are phosphoproteins (14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 15.Rochette-Egly C. Oulad-Abdelghani M. Staub A. Pfister V. Scheuer I. Chambon P. Gaub M.P. Mol. Endocrinol. 1995; 9: 860-871Crossref PubMed Google Scholar, 16.Adam-Stitah S. Penna L. Chambon P. Rochette-Egly C. J. Biol. Chem. 1999; 274: 18932-18941Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and their phosphorylation involves several kinases. RARα can be phosphorylated in its AF-1-containing B region by the cyclin-dependent kinase cdk7 (14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), which together with MAT1 and cyclin H forms the CAK complex that is found either free or as a component of the general transcription/DNA repair factor TFIIH (17.Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 18.Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (139) Google Scholar, 19.Rossignol M. Kolb-Cheynel I. Egly J.M. EMBO J. 1997; 16: 1628-1637Crossref PubMed Scopus (167) Google Scholar, 20.Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar). This phosphorylation, which results from an interaction with cdk7, is crucial for RARα transcriptional activity and modulates its ligand-induced degradation by the ubiquitin-proteasome pathway. 2E. Kopf, J. L. Plassat, V. Vivat, H. de Thé, P. Chambon, and C. Rochette-Egly, submitted for publication. 2E. Kopf, J. L. Plassat, V. Vivat, H. de Thé, P. Chambon, and C. Rochette-Egly, submitted for publication. RARα can also be phosphorylated by protein kinase A at a residue located in the ligand binding domain (15.Rochette-Egly C. Oulad-Abdelghani M. Staub A. Pfister V. Scheuer I. Chambon P. Gaub M.P. Mol. Endocrinol. 1995; 9: 860-871Crossref PubMed Google Scholar), and this phosphorylation is required for differentiation of mouse embryonal carcinoma F9 cells into parietal endoderm-like cells upon RA and cAMP treatment (13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Crossref PubMed Scopus (101) Google Scholar). Similarly, RXRα was found to be phosphorylated in its N-terminal A/B region and shown to be hyperphosphorylated in the same region by c-Jun N-terminal kinases upon UV activation (16.Adam-Stitah S. Penna L. Chambon P. Rochette-Egly C. J. Biol. Chem. 1999; 274: 18932-18941Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).Mutations of putative phosphorylation sites located in the AF-1 domain of mRARγ2 were found to prevent the RA-induced differentiation of F9 cells (13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Crossref PubMed Scopus (101) Google Scholar), thus indicating that RARγ2 phosphorylation in this domain could be required for this differentiation. Moreover, phosphorylation in the same domain was recently shown to be crucial for the ligand-induced degradation of RARγ by the ubiquitin-proteasome pathway.2 Because of our previous demonstration that RARα can be phosphorylated by cdk7 present within TFIIH, we assumed that RARγ could also be phosphorylated by TFIIH. In the present study, we demonstrate that the B region of the two major human or mouse RARγ isoforms, RARγ1 and RARγ2, are phosphorylated in a ligand-independent manner, by the cyclin H- and MAT1-dependent protein kinase cdk7. We also show that phosphorylation of RARγ is more efficient when cdk7 is present within TFIIH. In addition, we reveal the existence of multiple RARγ-TFIIH interactions that involve not only cdk7 but also additional subunits of TFIIH. Finally, phosphorylation of the A/B region was found to modulate RARγ-induced transcription in a response gene-dependent manner.DISCUSSIONIn this study, we have demonstrated that RARγ is phosphorylated in the N-terminal region that contains the activation function AF-1 (8.Nagpal S. Friant S. Nakshatri H. Chambon P. EMBO J. 1993; 12: 2349-2360Crossref PubMed Scopus (272) Google Scholar) and plays an essential role in RA-induced primitive endodermal differentiation of F9 cells (13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Crossref PubMed Scopus (101) Google Scholar). This phosphorylation, which involves two serine residues, is ligand-independent and appears to be most efficiently performed by the cyclin H-dependent kinase cdk7, a component of the general transcription/DNA repair factor TFIIH (17.Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 41.Svejstrup J.Q. Vichi P. Egly J.M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 42.Hoeijmakers J.H.J. Egly J.M. Vermeulen W. Curr. Opin. Genet. Dev. 1996; 6: 26-33Crossref PubMed Scopus (155) Google Scholar). Interestingly, this phosphorylation modulates the activity of AF-1 in a responsive gene-dependent manner.RARγ Is Phosphorylated by cdk7 in Its B and F RegionsRARγ is phosphorylated at two phosphorylation sites located in the B region. These sites are present in both the γ1 and γ2 isoforms and are conserved between human and mouse (21.Krust A. Kastner P. Petkovich M. Zelent A. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5310-5314Crossref PubMed Scopus (621) Google Scholar, 23.Kastner P. Krust A. Mendelsohn C. Garnier J.M. Zelent A. Leroy P. Staub A. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2700-2704Crossref PubMed Scopus (274) Google Scholar). They have been identified to serines 66 and 68 in RARγ2 and to serines 77 and 79 in RARγ1 (see Fig. 1). In this latter case, our data show that phosphorylation of serine 77 depends on that of serine 79. As previously reported for RARα (14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), we demonstrate that these sites are phosphorylated by cdk7, a cyclin H- and MAT1-dependent kinase. Indeed, overexpression of wild type cdk7, but not of cdk7 mutated at its ATP binding site or of any other cdk, results in a higher level of phosphorylation of the various RARs so far tested (Ref.14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). 3J. Bastien, S. Adam-Stitah, and C. Rochette-Egly, unpublished results. Moreover, similarly to RARα, the pattern of phosphorylation of RARγ appears to be independent of the phases of the cell cycle (Ref. 14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar).3As previously reported for RARα, RARγ is also phosphorylated in its F region. However, this phosphorylation concerns only mouse RARγ (either the γ1 or the γ2 isoforms) and not its human counterpart, due to the lack of conservation of this region between human and mouse (see Fig. 1). In contrast to phosphorylation of region B, no role has yet been found for this F region phosphorylation, either in RARα or in RARγ.RARγ Is More Efficiently Phosphorylated by TFIIH than by Free CAKCdk7 is associated with cyclin H and MAT1 in the CAK complex, and in the cell CAK is found either free or complexed with TFIIH, a general transcription factor also involved in DNA repair (17.Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 18.Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (139) Google Scholar, 19.Rossignol M. Kolb-Cheynel I. Egly J.M. EMBO J. 1997; 16: 1628-1637Crossref PubMed Scopus (167) Google Scholar, 20.Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar). Interestingly, we have shown that RARγ is more efficiently phosphorylated in vitro by cdk7 when included in TFIIH rather than in CAK, as previously reported for RARα (14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) and for the CTD of RNA polymerase II (19.Rossignol M. Kolb-Cheynel I. Egly J.M. EMBO J. 1997; 16: 1628-1637Crossref PubMed Scopus (167) Google Scholar, 41.Svejstrup J.Q. Vichi P. Egly J.M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 43.Makela T.P. Tassan J.P. Nigg E.A. Frutiger S. Hughes G.J. Weinberg R.A. Nature. 1994; 371: 254-257Crossref PubMed Scopus (232) Google Scholar, 44.Yankulov K. Yamashita K. Roy R. Egly J.M. Bentley D.L. J. Biol. Chem. 1995; 270: 23922-23925Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). That RARs are more efficiently phosphorylated by TFIIH than by CAK may result from RAR interactions not only with cdk7, as previously reported (Ref. 14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar and Fig. 5), but also with core subunits of TFIIH. Indeed, in coimmunoprecipitations experiments performed with insect cells coinfected with baculoviruses expressing different subunits of TFIIH and either RARγ or RARα, we found that both RARs interact not only with cdk7 in CAK and TFIIH but also with several subunits of the TFIIH core. Thus, these multiple interactions may account for more efficient phosphorylation by cdk7 within TFIIH than within free CAK. Note that other transcription factors such as p53 and E2F-1, which have been shown to be phosphorylated by cdk7, also interact with the core subunits of TFIIH (45.Leveillard T. Andera L. Bissonnette N. Schaeffer L. Bracco L. Egly J.M. Wasylyk B. EMBO J. 1996; 15: 1615-1624Crossref PubMed Scopus (138) Google Scholar, 46.Warbrick E. Curr. Biol. 1996; 6: 1057-1059Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 47.Qadri I. Conaway J.W. Conaway R.C. Schaack J. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10578-10583Crossref PubMed Scopus (129) Google Scholar).How cdk7 and the different TFIIH subunits interact with RARs remains to be investigated, but it is already clear from this and previous (14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) studies that the N-terminal A/B region is not mandatory for these interactions. Moreover, the interaction of TFIIH with RARs is not sensitive to deletion of the AF-2AD core/helix 12,3 which is involved in the coactivator binding surface of the ligand binding domain (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar). This is in accordance with our observation that the interaction of RARs with cdk7 and TFIIH is ligand-independent. Moreover, it suggests that the interaction between RARs and TFIIH involves another surface and is therefore mechanistically distinct from that described between RARs and coactivators (see the Introduction).Regulation of Transcription by RARγ through Phosphorylation of the AF-1 Domain by TFIIH-associated cdk7To investigate whether phosphorylation of the B region of RARs could modulate the ligand-induced activation of transcription, two reporter genes under the control of different responsive elements and promoters were tested: the natural mRARβ2 promoter, which contains a RARE with directly repeated motifs separated by 5 nucleotides (DR5), and the synthetic (TRE3)3tk promoter, which contains inverted (palindromic) repeated motifs. In both cases, RARγ activated transcription upon ligand binding. However, mutation of the phosphorylation sites located in the A/B region reduced transcription from the mRARβ2 promoter-based reporter gene, whereas it enhanced that from the (TRE3)3tk promoter-based reporter gene. The three-dimensional conformation of bound RXR/RAR heterodimers is most likely different on the two types of response elements. This may result in distinct steric conformations of the AF-1-activating domain and, therefore, in different interactions with putative AF-1 coactivators, which could be differentially modulated by phosphorylation. In this respect we note that interactions between coactivators and the AF-1-activating domain of either the estrogen receptor ERβ or the nuclear receptor SF-1 have been recently shown to be modulated by AF-1 phosphorylation (48.Hammer G.D Krylova I. Zhang Y. Darimont B.D. Simpson K. Weigel N. Ingraham H.A. Mol. Cell. 1999; 3: 521-526Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 49.Tremblay A. Tremblay G.B. Labrie F. Giguere V. Mol. Cell. 1999; 3: 513-519Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). In any event, such a possibility is in accordance with our previous report showing that phosphorylation of RARγ AF-1 is differentially required for RA-induced expression of target genes in F9 cells (13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Crossref PubMed Scopus (101) Google Scholar). Additionally, RAR phosphorylation may modulate the activity of TFIIH-associated cdk7 and/or regulate the enzymatic activity of some TFIIH subunits such as XPB and XPD that possess ATPase and helicase activity (18.Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (139) Google Scholar, 50.Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoeijmakers J.H. Chambon P. Egly J.M. Science. 1993; 260: 58-63Crossref PubMed Scopus (664) Google Scholar, 51.Schaeffer L. Moncollin V. Roy R. Staub A. Mezzina M. Sarasin A. Weeda G. Hoeijmakers J.H. Egly J.M. EMBO J. 1994; 13: 2388-2392Crossref PubMed Scopus (333) Google Scholar, 52.van Vuuren A.J. Appeldoorn E. Odijk H. Yasui A. Jaspers N.G. Bootsma D. Hoeijmakers J.H. EMBO J. 1993; 12: 3693-3701Crossref PubMed Scopus (140) Google Scholar, 53.Guzder S.N. Sung P. Bailly V. Prakash L. Prakash S. Nature. 1994; 369: 578-581Crossref PubMed Scopus (159) Google Scholar, 54.Guzder S.N. Qiu H. Sommers C.H. Sung P. Prakash L. Prakash S. Nature. 1994; 367: 91-94Crossref PubMed Scopus (121) Google Scholar) and are involved in distinct transcriptional steps (17.Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar).In conclusion, we have demonstrated that, as other transcriptional regulators such as p53 and E2-F (45.Leveillard T. Andera L. Bissonnette N. Schaeffer L. Bracco L. Egly J.M. Wasylyk B. EMBO J. 1996; 15: 1615-1624Crossref PubMed Scopus (138) Google Scholar, 46.Warbrick E. Curr. Biol. 1996; 6: 1057-1059Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 55.Vandel L. Kouzarides T. EMBO J. 1999; 18: 4280-4291Crossref PubMed Scopus (55) Google Scholar), RARs are targets for phosphorylation by cdk7 and interact with TFIIH. Thus, phosphorylation by TFIIH may be a general way of modulating the activity of transcriptional regulators. However, our present data also show that another proline-dependent kinase, p44MAPK, can also phosphorylate RARγ in vitro. Even though phosphorylation by this kinase is different from that achieved with cdk7 and overexpression of MAPK in COS cells does not affect the phosphorylation level and the transactivation properties of RARγ, our present data do not rule out the possibility that p44MAPKcould modulate RARγ activity in other cell types upon activation of the growth factor/MAPK cascade, as previously reported for ERα (56.Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki E. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1700) Google Scholar). The pleiotropic effects of retinoids are transduced by two nuclear receptor families, the retinoic acid receptors (RARs)1 and the retinoid X receptors (RXRs), that are ligand-dependent transregulators belonging to the nuclear receptor superfamily (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 2.Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (933) Google Scholar, 3.Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 4.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). RARs are activated by all-trans and 9-cis retinoic acid, whereas RXRs are activated by 9-cis retinoic acid only. There are three RAR (α, β, and γ) and three RXR (α, β, and γ) isotypes, and for each isotype there are at least two main isoforms that differ in their N-terminal region (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 5.Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 6.Chambon P. Semin. Cell Biol. 1994; 5: 115-125Crossref PubMed Scopus (499) Google Scholar). As do other members of the nuclear receptor superfamily, RARs and RXRs exhibit a conserved modular structure with six variably conserved regions (A to F) (Fig. 1) (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 5.Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). The N-terminal A/B region of RARs contains a ligand-independent transcriptional activation function, AF-1 (7.Nagpal S. Saunders M. Kastner P. Durand B. Nakshatri H. Chambon P. Cell. 1992; 70: 1007-1019Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 8.Nagpal S. Friant S. Nakshatri H. Chambon P. EMBO J. 1993; 12: 2349-2360Crossref PubMed Scopus (272) Google Scholar). Although the B regions of the three RAR isotypes are moderately conserved, their A regions are unrelated and differ for each isoform of a given RAR isotype (5.Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). The highly conserved C region encompasses the central DNA binding domain. The function of region F, if any, is unknown. Region E is more complex, as it contains the ligand binding domain, a dimerization interface, and the ligand-dependent transcriptional activation/repression domain AF-2 (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 9.Durand B. Saunders M. Gaudon C. Roy B. Losson R. Chambon P. EMBO J. 1994; 13: 5370-5382Crossref PubMed Scopus (315) Google Scholar). The activity of AF-2 is entirely dependent on the integrity of a conserved sequence referred to as the AF-2 AD core, located in α-helix 12 at the C-terminal end of the ligand binding domain. Ligand binding induces a major conformational change that includes helix 12 and creates a new surface for coactivator binding while corepressors are released, thus resulting in a transcriptional-competent nuclear receptor relayed to the transcriptional machinery and the chromatin template (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 10.Moras D. Gronemeyer H. Curr. Opin. Cell Biol. 1998; 10: 384-391Crossref PubMed Scopus (697) Google Scholar, 11.Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (509) Google Scholar, 12.Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (808) Google Scholar). The AF-2 and AF-1 activities synergize with each other in a response element- and promoter context-dependent manner (1.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2587) Google Scholar, 8.Nagpal S. Friant S. Nakshatri H. Chambon P. EMBO J. 1993; 12: 2349-2360Crossref PubMed Scopus (272) Google Scholar,13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Crossref PubMed Scopus (101) Google Scholar). RARs and RXRs are phosphoproteins (14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 15.Rochette-Egly C. Oulad-Abdelghani M. Staub A. Pfister V. Scheuer I. Chambon P. Gaub M.P. Mol. Endocrinol. 1995; 9: 860-871Crossref PubMed Google Scholar, 16.Adam-Stitah S. Penna L. Chambon P. Rochette-Egly C. J. Biol. Chem. 1999; 274: 18932-18941Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and their phosphorylation involves several kinases. RARα can be phosphorylated in its AF-1-containing B region by the cyclin-dependent kinase cdk7 (14.Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), which together with MAT1 and cyclin H forms the CAK complex that is found either free or as a component of the general transcription/DNA repair factor TFIIH (17.Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 18.Drapkin R. Le Roy G. Cho H. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6488-6493Crossref PubMed Scopus (139) Google Scholar, 19.Rossignol M. Kolb-Cheynel I. Egly J.M. EMBO J. 1997; 16: 1628-1637Crossref PubMed Scopus (167) Google Scholar, 20.Reardon J.T. Ge H. Gibbs E. Sancar A. Hurwitz J. Pan Z.Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6482-6487Crossref PubMed Scopus (99) Google Scholar). This phosphorylation, which results from an interaction with cdk7, is crucial for RARα transcriptional activity and modulates its ligand-induced degradation by the ubiquitin-proteasome pathway. 2E. Kopf, J. L. Plassat, V. Vivat, H. de Thé, P. Chambon, and C. Rochette-Egly, submitted for publication. 2E. Kopf, J. L. Plassat, V. Vivat, H. de Thé, P. Chambon, and C. Rochette-Egly, submitted for publication. RARα can also be phosphorylated by protein kinase A at a residue located in the ligand binding domain (15.Rochette-Egly C. Oulad-Abdelghani M. Staub A. Pfister V. Scheuer I. Chambon P. Gaub M.P. Mol. Endocrinol. 1995; 9: 860-871Crossref PubMed Google Scholar), and this phosphorylation is required for differentiation of mouse embryonal carcinoma F9 cells into parietal endoderm-like cells upon RA and cAMP treatment (13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Crossref PubMed Scopus (101) Google Scholar). Similarly, RXRα was found to be phosphorylated in its N-terminal A/B region and shown to be hyperphosphorylated in the same region by c-Jun N-terminal kinases upon UV activation (16.Adam-Stitah S. Penna L. Chambon P. Rochette-Egly C. J. Biol. Chem. 1999; 274: 18932-18941Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Mutations of putative phosphorylation sites located in the AF-1 domain of mRARγ2 were found to prevent the RA-induced differentiation of F9 cells (13.Taneja R. Rochette-Egly C. Plassat J.L. Penna L. Gaub M.P. Chambon P. EMBO J. 1997; 16: 6452-6465Cr