The Putative Cofactor TIF1α Is a Protein Kinase That Is Hyperphosphorylated upon Interaction with Liganded Nuclear Receptors

Abstract

Ligand-induced gene activation by nuclear receptors (NRs) is a complex process requiring dissociation of corepressors and recruitment of coactivators. The putative transcriptional intermediary factor TIF1α has been previously characterized as a nuclear protein that interacts directly with the AF-2 ligand-dependent activating domain present in the ligand-binding domain of numerous steroid and nonsteroid receptors, including the estrogen (ERα) and retinoid X (RXRα) receptors. We report here that TIF1α is both a phosphoprotein and a protein kinase. TIF1α coexpressed in COS-1 cells with RXRα or ERα is phosphorylated and becomes hyperphosphorylated upon ligand treatment. This hyperphosphorylation requires the binding of TIF1α to transcriptionally active NRs since it is prevented by mutations either in the core (α-helix 12 of the ligand-binding domain) of the AF-2 activating domains of RXRα and ERα or in the NR box of TIF1α that are known to prevent TIF1α-NR interactions. Thus, TIF1α is a phosphoprotein that undergoes ligand-dependent hyperphosphorylation as a consequence of nuclear receptor binding. We further show that purified recombinant TIF1α possesses intrinsic kinase activity and that, in addition to autophosphorylation, TIF1α selectively phosphorylates the transcription factors TFIIEα, TAFII28, and TAFII55 in vitro. These latter results raise the possibility that TIF1α may act, at least in part, by phosphorylating and modifying the activity of components of the transcriptional machinery. Ligand-induced gene activation by nuclear receptors (NRs) is a complex process requiring dissociation of corepressors and recruitment of coactivators. The putative transcriptional intermediary factor TIF1α has been previously characterized as a nuclear protein that interacts directly with the AF-2 ligand-dependent activating domain present in the ligand-binding domain of numerous steroid and nonsteroid receptors, including the estrogen (ERα) and retinoid X (RXRα) receptors. We report here that TIF1α is both a phosphoprotein and a protein kinase. TIF1α coexpressed in COS-1 cells with RXRα or ERα is phosphorylated and becomes hyperphosphorylated upon ligand treatment. This hyperphosphorylation requires the binding of TIF1α to transcriptionally active NRs since it is prevented by mutations either in the core (α-helix 12 of the ligand-binding domain) of the AF-2 activating domains of RXRα and ERα or in the NR box of TIF1α that are known to prevent TIF1α-NR interactions. Thus, TIF1α is a phosphoprotein that undergoes ligand-dependent hyperphosphorylation as a consequence of nuclear receptor binding. We further show that purified recombinant TIF1α possesses intrinsic kinase activity and that, in addition to autophosphorylation, TIF1α selectively phosphorylates the transcription factors TFIIEα, TAFII28, and TAFII55 in vitro. These latter results raise the possibility that TIF1α may act, at least in part, by phosphorylating and modifying the activity of components of the transcriptional machinery. Nuclear receptors (NRs) 1The abbreviations used are: NRs, nuclear receptors; LBD, ligand-binding domain; AD, activating domain; TF, transcription factor; RXR, retinoid X receptor; E2, estradiol; 9C-RA, 9-cis-retinoic acid; OHT, 4-hydroxytamoxifen; PIPES, 1,4-piperazinediethanesulfonic acid; NEB, nuclear extraction buffer; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; TIF, transcriptional intermediary factor; TBP, TATA-binding protein; TAF, TBP-associated protein; GST, glutathione S-transferase. represent a large family of ligand-inducible transcription factors that play numerous roles in the control of cell growth and differentiation, development, and homeostasis in response to small hydrophobic ligands, such as steroid and thyroid hormones, vitamin D, and retinoids (for reviews, see Refs.1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. 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A well conserved amphipathic α-helix (helix 12) referred to as the AF-2 AD core has been identified in the C-terminal part of the E region and shown to be an essential element of the ligand-inducible AF-2 function (see Refs. 1Mangelsdorf 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 (6165) Google Scholar, 2Gronemeyer H. Laudet V. Protein Profile. 1995; 2: 1173-1308PubMed Google Scholar, 3Chambon P. FASEB J. 1996; 10 (and references therein): 940-954Crossref PubMed Scopus (2622) Google Scholar, 4Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (942) Google Scholar). Upon ligand binding, this helix is thought to fold back over the LBD to generate transcriptionally active receptors (3Chambon P. FASEB J. 1996; 10 (and references therein): 940-954Crossref PubMed Scopus (2622) Google Scholar, 5Wurtz J.M. Bourguet W. Renaud J.P. 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Based on amino acid homologies, a typical TIF1 domain structure has been ascribed to members of this family, comprising an N-terminal region that contains a RBCC (RING finger-Bboxes-coiled coil) motif, a poorly conserved central region, and a C-terminal region that contains a PHD finger and a bromodomain (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar, 23Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). TIF1α was originally identified in a yeast genetic screen for mammalian proteins that can enhance the AF-2 activity of the retinoid X receptor (RXR) Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar) and was found to interact with the AF-2 of NRs in as well as in vitro Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar, E. Zechel C. Heery D. Heine M.J.S. Garnier J.M. Vivat V. Douarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: PubMed Scopus Google Scholar, S. C. H. V. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). TIF1α interacts with the proteins and (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar) and with the transcriptional domain which is present in of the finger proteins P. Douarin B. J.P. W. 1996; PubMed Scopus Google Scholar, R. Biol. Chem. 1997; Google Scholar). These results raise the possibility that TIF1α may play a role in the control of transcription at the level, involved both in the formation of transcriptionally and in activation to transcriptionally active (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar, R. Biol. Chem. 1997; Google Scholar). this that TIF1α is a phosphoprotein that undergoes hyperphosphorylation upon with nuclear receptors in TIF1α possesses intrinsic kinase activity for and of the transcription factors TFIIEα, TAFII28, and TAFII55 in vitro. These results that hyperphosphorylation of TIF1α upon with nuclear receptors, as well as of TFIIEα, TAFII28, and TAFII55 by TIF1α, be in the control of transcription by nuclear receptors. used in in COS-1 cells S. Chambon P. Full Text PDF PubMed Scopus Google Scholar) (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar). The by Douarin Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar) was by the of the expression with of the protein kinase A the and the COS-1 cells in the and for with 9C-RA, or in and in nuclear PIPES, and and at in nuclear extraction and of and and at the nuclear was to amino and and to Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). proteins and also used as and in the of and Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). ERα and RXRα and RXRα as S. Lutz Y. J.P. D. PubMed Scopus Google Scholar, C. Lutz Y. V. S. Chambon P. 1994; PubMed Scopus Google Scholar). Nuclear to and to The with by a and an cells with a TIF1α in a in growth at and and in The cells in a in and and as (15Fraser R.A. Rossignol M. Heard D.J. Egly J.M. Chambon P. J. Biol. Chem. 1997; 272: 7122-7126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The was and an which was and with The was directly an The was with and by and and with in the The and to and either or or by as of the acid was as (15Fraser R.A. Rossignol M. Heard D.J. Egly J.M. Chambon P. J. Biol. Chem. 1997; 272: 7122-7126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). the was with protein for at and the was with to protein in and protein was with a of of and in by and for kinase in E. and of recombinant TBP, and as V. J. A. Tora L. EMBO J. 1996; 15: PubMed Scopus Google Scholar). was in E. and purified by acid G. M. X. A. Tora L. Chambon P. J. 1995; 14: PubMed Scopus Google Scholar). is in E. was on a and with in the of in to and G. M. X. A. Tora L. Chambon P. J. 1995; 14: PubMed Scopus Google Scholar) purified by on and with with of purified in and of with or as at for and by the addition of and The phosphorylated proteins to and For in COS-1 cells in and in the for with as C. M. A. V. Chambon P. Mol. Endocrinol. 1995; PubMed Google Scholar). cell or nuclear to and and by and C. M. A. V. Chambon P. Mol. Endocrinol. 1995; PubMed Google Scholar). was to the gel by in A and proteins in A and by a of over a at in A The gel was for in kinase and and the was in the with of for at The gel was with and and The that TIF1α interacts with NRs in not stimulate the AF-2 activity of NRs in cells Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar), to the which TIF1α COS-1 cells with TIF1α in the or of RXRα or ERα and their and E2, The nuclear cells to and with a monoclonal A with a molecular of and which is in with the molecular of the TIF1α protein Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar), was in cells with TIF1α in the or of a of TIF1α a in in cells with either ERα 3 and or RXRα and Thus, TIF1α a upon with ERα or protein TIF1α, a protein Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar), be The COS-1 cells nuclear with in the or of the with both the and of TIF1α and whereas in the of the TIF1α was and Thus, the of TIF1α with the nuclear receptors RXRα and ERα to of this COS-1 cells TIF1α and RXRα with in the or of 9C-RA, and nuclear with to and the of RXRα and 9C-RA, TIF1α was phosphorylated D, and of RXRα and of the cells with hyperphosphorylation of TIF1α, in a of D, and these results that the NR-induced TIF1α as it is by and is by an the hyperphosphorylation of TIF1α was for that AF-2 with ERα in the of the is known to prevent ERα AF-2 activity in mammalian and yeast cells M. D. Chambon P. EMBO J. 1990; PubMed Scopus Google D. Losson R. J.M. Y. Chambon P. PubMed Scopus Google Scholar) and to the between the LBD of ERα and TIF1α Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). cells with TIF1α and to of TIF1α and the by and This that hyperphosphorylation of TIF1α requires the transcriptionally active of the ERα that is required for with has been previously in vitro and in yeast Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar) that the ligand-dependent of NRs with TIF1α requires the of the core of the AF-2 AD (α-helix this was required for TIF1α with TIF1α and RXRα either the AF-2 AD core or amino acid in the conserved hydrophobic and RXRα Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar, E. Zechel C. Heery D. Heine M.J.S. Garnier J.M. Vivat V. Douarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: PubMed Scopus Google Scholar, B. M. C. B. Losson R. Chambon P. EMBO J. 1994; 13: PubMed Scopus Google Scholar). to RXRα and the RXRα and and the AF-2 AD core to the upon amino acid mutations in the AF-2 AD core of ERα that prevent the with TIF1α (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar) also the of TIF1α with and These which are in with the interactions both in yeast and in vitro Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar, E. Zechel C. Heery D. Heine M.J.S. Garnier J.M. Vivat V. Douarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: PubMed Scopus Google Scholar), that TIF1α hyperphosphorylation by RXRα or ERα requires the of the AF-2 AD core (α-helix 12 of the The and AF-2 AD core-dependent TIF1α-NR is also known to the TIF1α NR box to (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar), and mutations in this NR box the binding of TIF1α to nuclear receptors (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar). the TIF1α NR box was required for NR-induced of TIF1α, and TIF1α and RXRα expression with RXRα in COS-1 cells with and of the NR box TIF1α hyperphosphorylation and The results cells with NR box TIF1α and ERα and with not Thus, TIF1α hyperphosphorylation requires an NR these results that the of TIF1α, which in the of RXRα and the as previously shown to be for the binding of TIF1α to the LBD of the of the kinase for TIF1α recombinant a expression in cells and purified it to by by (see a of and purified TIF1α was in vitro kinase TIF1α was phosphorylated the addition of other factors or that TIF1α have an activity and the possibility that this kinase activity be to a an was was three monoclonal and a TIF1α to the was with with an of the The the TIF1α, and the to an in vitro kinase and by and the and TIF1α the three and in the kinase was phosphorylated and Thus, three and the ability to the possibility that a kinase associated with TIF1α these three was by and be The was for activity in an kinase protein in to was phosphorylated the possibility that the of TIF1α be to a kinase. was in with or cells not the of TIF1α not possess well conserved domains typical of the kinase be conserved at amino of the TIF1α amino acid Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). TIF1α is in and Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar), further be required to the phosphorylated amino and to the kinase We TIF1α other The proteins to be known to interact directly with TIF1α, such as and (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar, 23Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). of the recombinant proteins phosphorylated by purified TIF1α in vitro kinase not TIF1α ERα and RXRα in a COS-1 cells with TIF1α and either ERα or RXRα with with or The of ERα was by 3 and this was not by of either TIF1α and or TIF1α in the NR box 3 and TIF1α not the of of in the of 3 TIF1α is a putative transcriptional intermediary factor for nuclear receptors (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar, 23Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). the ability of TIF1α to recombinant transcription factors such as TFIIEα, TBP, TAFII28, and TAFII55 V. J. A. Tora L. EMBO J. 1996; 15: PubMed Scopus Google Scholar, G. M. X. A. Tora L. Chambon P. J. 1995; 14: PubMed Scopus Google Scholar) was in vitro not these factors, and the TBP-associated factors and TAFII55 phosphorylated by recombinant and stable of TIF1α with these factors be found not as previously with other known components of the transcriptional Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar). TIF1α was originally identified as a transcriptional intermediary factor on the basis of direct ligand-dependent with NRs in in Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (577) Google Scholar, E. Zechel C. Heery D. Heine M.J.S. Garnier J.M. Vivat V. Douarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: PubMed Scopus Google Scholar). have the that TIF1α may at least in part, transcription by NRs (22Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (475) Google Scholar). We here that TIF1α undergoes hyperphosphorylation that is on direct with a nuclear receptor upon binding of the We also that TIF1α possesses an intrinsic kinase activity as it and phosphorylates transcription factors such as TFIIEα, TAFII28, and TAFII55 in vitro. Thus, TIF1α is both a phosphoprotein and a protein kinase.