Phosphorylation of TCF Proteins by Homeodomain-interacting Protein Kinase 2
2011; Elsevier BV; Volume: 286; Issue: 14 Linguagem: Inglês
10.1074/jbc.m110.185280
ISSN1083-351X
AutoresHiroki Hikasa, Sergei Y. Sokol,
Tópico(s)Developmental Biology and Gene Regulation
ResumoWnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos. Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos. IntroductionWnt signaling is an essential embryonic pathway that regulates cell fate determination, cell proliferation, and cell polarity. The Wnt pathway leads to the stabilization of β-catenin, which associates with TCF proteins to activate target genes (1MacDonald B.T. Tamai K. He X. Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4057) Google Scholar, 2Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4430) Google Scholar). Whereas the function of β-catenin in embryonic axis determination and Wnt signaling has been firmly established (3Grigoryan T. Wend P. Klaus A. Birchmeier W. Genes Dev. 2008; 22: 2308-2341Crossref PubMed Scopus (461) Google Scholar, 4Heasman J. Kofron M. Wylie C. Dev. Biol. 2000; 222: 124-134Crossref PubMed Scopus (460) Google Scholar, 5Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4180) Google Scholar), genetic studies of TCF proteins reveal their diverse and complex roles in development (6van Genderen C. Okamura R.M. Fariñas I. Quo R.G. Parslow T.G. Bruhn L. Grosschedl R. Genes Dev. 1994; 8: 2691-2703Crossref PubMed Scopus (814) Google Scholar, 7Arce L. Yokoyama N.N. Waterman M.L. Oncogene. 2006; 25: 7492-7504Crossref PubMed Scopus (332) Google Scholar, 8Galceran J. Fariñas I. Depew M.J. Clevers H. Grosschedl R. Genes Dev. 1999; 13: 709-717Crossref PubMed Scopus (399) Google Scholar, 9Standley H.J. Destrée O. Kofron M. Wylie C. Heasman J. Dev. Biol. 2006; 289: 318-328Crossref PubMed Scopus (43) Google Scholar, 10Liu F. van den Broek O. Destrée O. Hoppler S. Development. 2005; 132: 5375-5385Crossref PubMed Scopus (91) Google Scholar, 11Roël G. Hamilton F.S. Gent Y. Bain A.A. Destrée O. Hoppler S. Curr. Biol. 2002; 12: 1941-1945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In a commonly accepted canonical model, TCFs bind Groucho/TLE corepressor proteins and inhibit target genes in the absence of a Wnt signal but associate with β-catenin and convert into activators after cell stimulation by Wnt proteins (12Daniels D.L. Weis W.I. Nat. Struct. Mol. Biol. 2005; 12: 364-371Crossref PubMed Scopus (423) Google Scholar, 13van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Loureiro J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 14Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2579) Google Scholar). In organisms, which possess a single TCF gene, such as Caenorhabditis elegans (POP-1) or Drosophila (pangolin/dTCF), TCF proteins play both negative and positive roles (15Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (444) Google Scholar, 16Cavallo R.A. Cox R.T. Moline M.M. Roose J. Polevoy G.A. Clevers H. Peifer M. Bejsovec A. Nature. 1998; 395: 604-608Crossref PubMed Scopus (591) Google Scholar, 17Phillips B.T. Kimble J. Dev. Cell. 2009; 17: 27-34Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). By contrast, vertebrates carry four conserved TCF homologues, TCF1, LEF1, TCF3, and TCF4, which appear to have distinct functions and control different sets of targets at different times during development (7Arce L. Yokoyama N.N. Waterman M.L. Oncogene. 2006; 25: 7492-7504Crossref PubMed Scopus (332) Google Scholar). Despite the important roles for TCF proteins in the control of gene expression during development and disease (1MacDonald B.T. Tamai K. He X. Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4057) Google Scholar, 2Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4430) Google Scholar, 7Arce L. Yokoyama N.N. Waterman M.L. Oncogene. 2006; 25: 7492-7504Crossref PubMed Scopus (332) Google Scholar), the mechanisms of their regulation are still poorly understood.Accumulating evidence shows that TCF proteins can be phosphorylated in response to Wnt proteins, raising the question whether this phoshorylation is important for determining the outcome of signaling. For example, phosphorylation of Xenopus TCF3 by casein kinase 1, a critical player in Wnt signaling (18Price M.A. Genes Dev. 2006; 20: 399-410Crossref PubMed Scopus (208) Google Scholar, 19Peters J.M. McKay R.M. McKay J.P. Graff J.M. Nature. 1999; 401: 345-350Crossref PubMed Scopus (382) Google Scholar, 20Davidson G. Wu W. Shen J. Bilic J. Fenger U. Stannek P. Glinka A. Niehrs C. Nature. 2005; 438: 867-872Crossref PubMed Scopus (460) Google Scholar) was proposed to stimulate β-catenin binding (21Lee E. Salic A. Kirschner M.W. J. Cell Biol. 2001; 154: 983-993Crossref PubMed Scopus (126) Google Scholar). In C. elegans, the protein kinase LIT-1 triggers POP-1/TCF phosphorylation, leading to its nuclear export that is required to promote the endodermal fate during Wnt signaling (22Lin R. Thompson S. Priess J.R. Cell. 1995; 83: 599-609Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 23Lo M.C. Gay F. Odom R. Shi Y. Lin R. Cell. 2004; 117: 95-106Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 24Rocheleau C.E. Downs W.D. Lin R. Wittmann C. Bei Y. Cha Y.H. Ali M. Priess J.R. Mello C.C. Cell. 1997; 90: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 25Rocheleau C.E. Yasuda J. Shin T.H. Lin R. Sawa H. Okano H. Priess J.R. Davis R.J. Mello C.C. Cell. 1999; 97: 717-726Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Similarly, vertebrate TCF proteins LEF1 and TCF4 can be phosphorylated in cultured cells by Nlk (Nemo-like kinase), a mammalian homologue of LIT-1 (26Kaletta T. Schnabel H. Schnabel R. Nature. 1997; 390: 294-298Crossref PubMed Scopus (132) Google Scholar, 27Meneghini M.D. Ishitani T. Carter J.C. Hisamoto N. Ninomiya-Tsuji J. Thorpe C.J. Hamill D.R. Matsumoto K. Bowerman B. Nature. 1999; 399: 793-797Crossref PubMed Scopus (234) Google Scholar, 28Smit L. Baas A. Kuipers J. Korswagen H. van de Wetering M. Clevers H. J. Biol. Chem. 2004; 279: 17232-17240Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29Ishitani T. Ninomiya-Tsuji J. Nagai S. Nishita M. Meneghini M. Barker N. Waterman M. Bowerman B. Clevers H. Shibuya H. Matsumoto K. Nature. 1999; 399: 798-802Crossref PubMed Scopus (515) Google Scholar, 30Ishitani T. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell Biol. 2003; 23: 1379-1389Crossref PubMed Scopus (183) Google Scholar), but the in vivo significance of this phosphorylation has not been established.Another family of nuclear protein kinases that have been implicated in Wnt signaling and could play a role in TCF regulation are homeodomain-interacting protein kinases (HIPK1–4) (31Kim Y.H. Choi C.Y. Lee S.J. Conti M.A. Kim Y. J. Biol. Chem. 1998; 273: 25875-25879Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). HIPK2 is expressed in multiple mouse embryonic tissues, including the brain, the heart, the kidney, and the muscle (32Pierantoni G.M. Bulfone A. Pentimalli F. Fedele M. Iuliano R. Santoro M. Chiariotti L. Ballabio A. Fusco A. Biochem. Biophys. Res. Commun. 2002; 290: 942-947Crossref PubMed Scopus (45) Google Scholar), and functions in transcriptional regulation, cell growth, and apoptosis (33Kondo S. Lu Y. Debbas M. Lin A.W. Sarosi I. Itie A. Wakeham A. Tuan J. Saris C. Elliott G. Ma W. Benchimol S. Lowe S.W. Mak T.W. Thukral S.K. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 5431-5436Crossref PubMed Scopus (54) Google Scholar, 34Doxakis E. Huang E.J. Davies A.M. Curr. Biol. 2004; 14: 1761-1765Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), presumably by activating p53 (35D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (566) Google Scholar, 36Hofmann T.G. Möller A. Sirma H. Zentgraf H. Taya Y. Dröge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (492) Google Scholar, 37Dauth I. Krüger J. Hofmann T.G. Cancer Res. 2007; 67: 2274-2279Crossref PubMed Scopus (66) Google Scholar) or c-Jun N-terminal kinase (38Hofmann T.G. Stollberg N. Schmitz M.L. Will H. Cancer Res. 2003; 63: 8271-8277PubMed Google Scholar). Embryos from mice lacking both HIPK1 and HIPK2 genes exhibit severe exencephaly with anterior neural tissue overgrowth and die between embryonic days 9.5 and 12.5 (39Isono K. Nemoto K. Li Y. Takada Y. Suzuki R. Katsuki M. Nakagawara A. Koseki H. Mol. Cell Biol. 2006; 26: 2758-2771Crossref PubMed Scopus (91) Google Scholar). HIPK2-mediated phosphorylation promotes proteasome-dependent degradation of C-terminal binding protein (41Zhang Q. Yoshimatsu Y. Hildebrand J. Frisch S.M. Goodman R.H. Cell. 2003; 115: 177-186Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and attenuates Groucho repressive activity (40Choi C.Y. Kim Y.H. Kim Y.O. Park S.J. Kim E.A. Riemenschneider W. Gajewski K. Schulz R.A. Kim Y. J. Biol. Chem. 2005; 280: 21427-21436Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The HIPK2-Nlk complex was demonstrated to phosphorylate and degrade c-Myb in response to Wnt1 (42Kanei-Ishii C. Ninomiya-Tsuji J. Tanikawa J. Nomura T. Ishitani T. Kishida S. Kokura K. Kurahashi T. Ichikawa-Iwata E. Kim Y. Matsumoto K. Ishii S. Genes Dev. 2004; 18: 816-829Crossref PubMed Scopus (153) Google Scholar). Other studies reported both positive and negative effects of HIPK proteins in Wnt/β-catenin signaling in mouse embryo fibroblasts (43Wei G. Ku S. Ma G.K. Saito S. Tang A.A. Zhang J. Mao J.H. Appella E. Balmain A. Huang E.J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13040-13045Crossref PubMed Scopus (114) Google Scholar, 44Kim E.A. Kim J.E. Sung K.S. Choi D.W. Lee B.J. Choi C.Y. Biochem. Biophys. Res. Commun. 2010; 394: 966-971Crossref PubMed Scopus (31) Google Scholar), Drosophila and Xenopus embryos (45Lee W. Swarup S. Chen J. Ishitani T. Verheyen E.M. Development. 2009; 136: 241-251Crossref PubMed Scopus (63) Google Scholar, 46Louie S.H. Yang X.Y. Conrad W.H. Muster J. Angers S. Moon R.T. Cheyette B.N. PLoS ONE. 2009; 4: e4310Crossref PubMed Scopus (29) Google Scholar), but the underlying mechanisms have not been fully elucidated.We have recently discovered that TCF3 is phosphorylated by HIPK2 in response to Wnt8 stimulation and identified the relevant phosphorylation sites critical for its function (47Hikasa H. Ezan J. Itoh K. Li X. Klymkowsky M.W. Sokol S.Y. Dev. Cell. 2010; 19: 521-532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Based on the conservation of some of these phosphorylation sites in LEF1, TCF3, and TCF4 but not in TCF1, we hypothesize that HIPK2 is involved in the phosphorylation of different TCF proteins. To test this hypothesis, we examined the phosphorylation state of TCF family proteins and observed a similar regulation of LEF1 and TCF4, but not TCF1, by Wnt/HIPK2-dependent phosphorylation. Our data indicate that the physiological role for this phosphorylation is to decrease TCF binding to target promoters. Moreover, we find that this phosphorylation leads to the replacement of the TCF3 repressor with the TCF1 activator, revealing a novel "TCF switch" mechanism for transcriptional activation.DISCUSSIONIn this work, we analyzed the mechanism of TCF protein regulation by HIPK2 in Xenopus embryos. We find that Wnt stimulation or overexpression of HIPK2 cause phosphorylation of LEF-1, TCF4, and TCF3. This phosphorylation leads to the dissociation of TCF proteins from a target promoter and promotes gene target activation (in case of TCF3) or transcriptional repression (in case of LEF1). Because TCF proteins are the most downstream components of the signal transduction pathway from the cell surface to the nucleus, this regulation should be no less important than the control of β-catenin stability. Future studies will evaluate whether this phosphorylation is a diagnostic marker for cancers and whether it can serve as a basis of new drug screens.The observed mechanism seems to be similar to the phosphorylation of POP1/TCF by the MOM-4/LIT1 kinase in C. elegans and mammalian TCF4 by the Wnt1/TGFβ-activated kinase/Nlk cascade in HEK293T cells, for which the upstream components are unknown (22Lin R. Thompson S. Priess J.R. Cell. 1995; 83: 599-609Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 25Rocheleau C.E. Yasuda J. Shin T.H. Lin R. Sawa H. Okano H. Priess J.R. Davis R.J. Mello C.C. Cell. 1999; 97: 717-726Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 27Meneghini M.D. Ishitani T. Carter J.C. Hisamoto N. Ninomiya-Tsuji J. Thorpe C.J. Hamill D.R. Matsumoto K. Bowerman B. Nature. 1999; 399: 793-797Crossref PubMed Scopus (234) Google Scholar, 28Smit L. Baas A. Kuipers J. Korswagen H. van de Wetering M. Clevers H. J. Biol. Chem. 2004; 279: 17232-17240Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29Ishitani T. Ninomiya-Tsuji J. Nagai S. Nishita M. Meneghini M. Barker N. Waterman M. Bowerman B. Clevers H. Shibuya H. Matsumoto K. Nature. 1999; 399: 798-802Crossref PubMed Scopus (515) Google Scholar, 89Shin T.H. Yasuda J. Rocheleau C.E. Lin R. Soto M. Bei Y. Davis R.J. Mello C.C. Mol. Cell. 1999; 4: 275-280Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Previous study reported that LEF1 is phosphorylated in response to Wnt5a/CamKII signaling (72Ishitani T. Kishida S. Hyodo-Miura J. Ueno N. Yasuda J. Waterman M. Shibuya H. Moon R.T. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell Biol. 2003; 23: 131-139Crossref PubMed Scopus (469) Google Scholar), whereas we observed that TCF3 is phosphorylated by Wnt8/HIPK2 signaling (47Hikasa H. Ezan J. Itoh K. Li X. Klymkowsky M.W. Sokol S.Y. Dev. Cell. 2010; 19: 521-532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In this study, we found that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, but not β-catenin, indicating that TCF phosphorylation and β-catenin stabilization share the same upstream regulators. Interestingly, both Nlk and HIPK2 were reported to regulate c-Myb degradation in response to Wnt1 (42Kanei-Ishii C. Ninomiya-Tsuji J. Tanikawa J. Nomura T. Ishitani T. Kishida S. Kokura K. Kurahashi T. Ichikawa-Iwata E. Kim Y. Matsumoto K. Ishii S. Genes Dev. 2004; 18: 816-829Crossref PubMed Scopus (153) Google Scholar). Further experiments are required to better understand the relationship between the TGFβ-activated kinase/Nlk- and the canonical Wnt/HIPK2-mediated TCF phosphorylation.Recent studies reached diverse conclusions regarding the role of HIPK in Wnt signaling. HIPK homologues were reported to suppress Wnt target gene expression in several experimental models (43Wei G. Ku S. Ma G.K. Saito S. Tang A.A. Zhang J. Mao J.H. Appella E. Balmain A. Huang E.J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13040-13045Crossref PubMed Scopus (114) Google Scholar, 44Kim E.A. Kim J.E. Sung K.S. Choi D.W. Lee B.J. Choi C.Y. Biochem. Biophys. Res. Commun. 2010; 394: 966-971Crossref PubMed Scopus (31) Google Scholar, 46Louie S.H. Yang X.Y. Conrad W.H. Muster J. Angers S. Moon R.T. Cheyette B.N. PLoS ONE. 2009; 4: e4310Crossref PubMed Scopus (29) Google Scholar), but positively regulate signaling in other models (45Lee W. Swarup S. Chen J. Ishitani T. Verheyen E.M. Development. 2009; 136: 241-251Crossref PubMed Scopus (63) Google Scholar, 47Hikasa H. Ezan J. Itoh K. Li X. Klymkowsky M.W. Sokol S.Y. Dev. Cell. 2010; 19: 521-532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). To explain the context-dependent function of HIPK proteins, one needs to consider that different TCF proteins are expressed in the spatially and temporally restricted fashion and have diverse roles in early development (9Standley H.J. Destrée O. Kofron M. Wylie C. Heasman J. Dev. Biol. 2006; 289: 318-328Crossref PubMed Scopus (43) Google Scholar, 10Liu F. van den Broek O. Destrée O. Hoppler S. Development. 2005; 132: 5375-5385Crossref PubMed Scopus (91) Google Scholar, 11Roël G. Hamilton F.S. Gent Y. Bain A.A. Destrée O. Hoppler S. Curr. Biol. 2002; 12: 1941-1945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). According to our model, HIPK2 functions as a positive or negative regulator of Wnt signaling, depending on the functional properties of TCF proteins that are present in the embryonic tissue. Specifically, HIPK2 would inhibit the pathway when an activator type TCF, such as LEF1, is phosphorylated, but would activate it when phosphorylating the repressive form of TCF (TCF3). Indeed, HIPK2 stimulates a Vent2 reporter by phosphorylating TCF3 (47Hikasa H. Ezan J. Itoh K. Li X. Klymkowsky M.W. Sokol S.Y. Dev. Cell. 2010; 19: 521-532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) but inhibits LEF-1-dependent reporter activation by phosphorylating LEF1 (Figs. 3D and 4A). This observation is consistent with the study, in which the phosphorylation of the P3 site by Nlk was proposed to inhibit LEF1 activity (30Ishitani T. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell Biol. 2003; 23: 1379-1389Crossref PubMed Scopus (183) Google Scholar). These findings are strongly supported by our data revealing the dissociation of different TCF proteins from the Vent2 promoter (Fig. 4). There is no effect of HIPK2 on TCF1, consistent with the lack of the P2/P3/P4 phosphorylation sites in this gene. Together, our results provide a likely mechanism for the context-dependent effects of HIPK2 on Wnt signaling.The regulatory mechanism for Wnt-dependent TCF phosphorylation differs significantly from the commonly accepted model of β-catenin/TCF coactivation of Wnt target genes, yet it has the same upstream regulators. It is unclear under which circumstances one branch is favored versus the other and whether the two branches might operate simultaneously. So far, we were unable to detect a change in HIPK2 enzymatic activity in embryos after Wnt stimulation (data not shown). We observed that at high doses of the Wnt signal, the Vent2 reporter was activated to a higher degree than when the TCF3 repression was removed by TCF3 ΜO or when the TCF-binding site has been mutated (Fig. 5A and data not shown). This suggests the existence of an activation mechanism in addition to the derepression mechanism (Fig. 5D). This model is supported by our finding that upon HIPK2-mediated phosphorylation, TCF3 is replaced by TCF1 at the target promoter. Given that both TCF3 and TCF1 are co-expressed during gastrulation and function antagonistically (9Standley H.J. Destrée O. Kofron M. Wylie C. Heasman J. Dev. Biol. 2006; 289: 318-328Crossref PubMed Scopus (43) Google Scholar, 10Liu F. van den Broek O. Destrée O. Hoppler S. Development. 2005; 132: 5375-5385Crossref PubMed Scopus (91) Google Scholar, 47Hikasa H. Ezan J. Itoh K. Li X. Klymkowsky M.W. Sokol S.Y. Dev. Cell. 2010; 19: 521-532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), this mechanism is likely to operate during anteroposterior embryonic development. IntroductionWnt signaling is an essential embryonic pathway that regulates cell fate determination, cell proliferation, and cell polarity. The Wnt pathway leads to the stabilization of β-catenin, which associates with TCF proteins to activate target genes (1MacDonald B.T. Tamai K. He X. Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4057) Google Scholar, 2Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4430) Google Scholar). Whereas the function of β-catenin in embryonic axis determination and Wnt signaling has been firmly established (3Grigoryan T. Wend P. Klaus A. Birchmeier W. Genes Dev. 2008; 22: 2308-2341Crossref PubMed Scopus (461) Google Scholar, 4Heasman J. Kofron M. Wylie C. Dev. Biol. 2000; 222: 124-134Crossref PubMed Scopus (460) Google Scholar, 5Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4180) Google Scholar), genetic studies of TCF proteins reveal their diverse and complex roles in development (6van Genderen C. Okamura R.M. Fariñas I. Quo R.G. Parslow T.G. Bruhn L. Grosschedl R. Genes Dev. 1994; 8: 2691-2703Crossref PubMed Scopus (814) Google Scholar, 7Arce L. Yokoyama N.N. Waterman M.L. Oncogene. 2006; 25: 7492-7504Crossref PubMed Scopus (332) Google Scholar, 8Galceran J. Fariñas I. Depew M.J. Clevers H. Grosschedl R. Genes Dev. 1999; 13: 709-717Crossref PubMed Scopus (399) Google Scholar, 9Standley H.J. Destrée O. Kofron M. Wylie C. Heasman J. Dev. Biol. 2006; 289: 318-328Crossref PubMed Scopus (43) Google Scholar, 10Liu F. van den Broek O. Destrée O. Hoppler S. Development. 2005; 132: 5375-5385Crossref PubMed Scopus (91) Google Scholar, 11Roël G. Hamilton F.S. Gent Y. Bain A.A. Destrée O. Hoppler S. Curr. Biol. 2002; 12: 1941-1945Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In a commonly accepted canonical model, TCFs bind Groucho/TLE corepressor proteins and inhibit target genes in the absence of a Wnt signal but associate with β-catenin and convert into activators after cell stimulation by Wnt proteins (12Daniels D.L. Weis W.I. Nat. Struct. Mol. Biol. 2005; 12: 364-371Crossref PubMed Scopus (423) Google Scholar, 13van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Loureiro J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 14Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2579) Google Scholar). In organisms, which possess a single TCF gene, such as Caenorhabditis elegans (POP-1) or Drosophila (pangolin/dTCF), TCF proteins play both negative and positive roles (15Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (444) Google Scholar, 16Cavallo R.A. Cox R.T. Moline M.M. Roose J. Polevoy G.A. Clevers H. Peifer M. Bejsovec A. Nature. 1998; 395: 604-608Crossref PubMed Scopus (591) Google Scholar, 17Phillips B.T. Kimble J. Dev. Cell. 2009; 17: 27-34Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). By contrast, vertebrates carry four conserved TCF homologues, TCF1, LEF1, TCF3, and TCF4, which appear to have distinct functions and control different sets of targets at different times during development (7Arce L. Yokoyama N.N. Waterman M.L. Oncogene. 2006; 25: 7492-7504Crossref PubMed Scopus (332) Google Scholar). Despite the important roles for TCF proteins in the control of gene expression during development and disease (1MacDonald B.T. Tamai K. He X. Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4057) Google Scholar, 2Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4430) Google Scholar, 7Arce L. Yokoyama N.N. Waterman M.L. Oncogene. 2006; 25: 7492-7504Crossref PubMed Scopus (332) Google Scholar), the mechanisms of their regulation are still poorly understood.Accumulating evidence shows that TCF proteins can be phosphorylated in response to Wnt proteins, raising the question whether this phoshorylation is important for determining the outcome of signaling. For example, phosphorylation of Xenopus TCF3 by casein kinase 1, a critical player in Wnt signaling (18Price M.A. Genes Dev. 2006; 20: 399-410Crossref PubMed Scopus (208) Google Scholar, 19Peters J.M. McKay R.M. McKay J.P. Graff J.M. Nature. 1999; 401: 345-350Crossref PubMed Scopus (382) Google Scholar, 20Davidson G. Wu W. Shen J. Bilic J. Fenger U. Stannek P. Glinka A. Niehrs C. Nature. 2005; 438: 867-872Crossref PubMed Scopus (460) Google Scholar) was proposed to stimulate β-catenin binding (21Lee E. Salic A. Kirschner M.W. J. Cell Biol. 2001; 154: 983-993Crossref PubMed Scopus (126) Google Scholar). 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PLoS ONE. 2009; 4: e4310Crossref PubMed Scopus (29) Google Scholar), but the underlying mechanisms have not been fully elucidated.We have recently discovered that TCF3 is phosphorylated by HIPK2 in response to Wnt8 stimulation and identified the relevant phosphorylation sites critical for its function (47Hikasa H. Ezan J. Itoh K. Li X. Klymkowsky M.W. Sokol S.Y. Dev. Cell. 2010; 19: 521-532Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Based on the conservation of some of these phosphorylation sites in LEF1, TCF3, and TCF4 but not in TCF1, we hypothesize that HIPK2 is involved in the phosphorylation of different TCF proteins. To test this hypothesis, we examined the phosphorylation state of TCF family proteins and observed a similar regulation of LEF1 and TCF4, but not TCF1, by Wnt/HIPK2-dependent phosphorylation. Our data indicate that the physiological role for this phosphorylation is to decrease TCF binding to target promoters. Moreover, we find that this phosphorylation leads to the replacement of the TCF3 repressor with the TCF1 activator, revealing a novel "TCF switch" mechanism for transcriptional activation.
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