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Association of the TLX-2 Homeodomain and 14-3-3η Signaling Proteins

1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês

10.1074/jbc.273.39.25356

1083-351X

Shao Jun Tang, T C Suen, Roderick R. McInnes, Manuel Buchwald,

Signaling Pathways in Disease

Homeodomain proteins play important roles in various developmental processes, and their functions are modulated by polypeptide cofactors. Here we report that both in vitroand in vivo, 14-3-3η is associated with the TLX-2 homeodomain transcription factor that is required for mouse embryogenesis. Expression of 14-3-3η shifts the predominant localization of TLX-2 in COS cells from the cytoplasm to the nucleus.Tlx-2 and 14-3-3η are expressed in the developing peripheral nervous system with spatially and temporally overlapping patterns, and they are also coexpressed in PC12 cells. Increased expression of either gene by transfection considerably inhibited nerve growth factor-induced neurite outgrowth of PC12 cells, and cotransfection of both genes led to a synergistic effect of suppression. These findings define 14-3-3η as a functional modulator of the TLX-2 homeodomain transcription factor and suggest that thein vivo function of TLX-2 in neural differentiation is likely regulated by signaling mediated by 14-3-3η. Homeodomain proteins play important roles in various developmental processes, and their functions are modulated by polypeptide cofactors. Here we report that both in vitroand in vivo, 14-3-3η is associated with the TLX-2 homeodomain transcription factor that is required for mouse embryogenesis. Expression of 14-3-3η shifts the predominant localization of TLX-2 in COS cells from the cytoplasm to the nucleus.Tlx-2 and 14-3-3η are expressed in the developing peripheral nervous system with spatially and temporally overlapping patterns, and they are also coexpressed in PC12 cells. Increased expression of either gene by transfection considerably inhibited nerve growth factor-induced neurite outgrowth of PC12 cells, and cotransfection of both genes led to a synergistic effect of suppression. These findings define 14-3-3η as a functional modulator of the TLX-2 homeodomain transcription factor and suggest that thein vivo function of TLX-2 in neural differentiation is likely regulated by signaling mediated by 14-3-3η. peripheral nervous system glutathione S-transferase phosphate-buffered saline fluorescein isothiocyanate nerve growth factor 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside. Homeobox genes encode an evolutionarily conserved superfamily of transcription factors that play vital roles in various aspects of development (1Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1730) Google Scholar). A number of cofactors, including homeoproteins (2Xue D. Tu Y. Chalfie M. Science. 1993; 261: 1324-1328Crossref PubMed Scopus (181) Google Scholar, 3Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. Cell. 1994; 78: 603-615Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 4Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar), specific transcription factors (5Grueneberg D. Natesan S. Alexandre C. Gilman M. Science. 1992; 257: 1089-1095Crossref PubMed Scopus (256) Google Scholar, 6Guichet A. Copeland J.W.R. Erdelyi M. Hlousek D. Zavorszky P. Ho J. Brown S. Percival-Smith A. Krause H.M. Ephrussi A. Nature. 1997; 385: 548-552Crossref PubMed Scopus (158) Google Scholar, 7Yu Y. Li W. Yussa M. Han W. Perrimon N. Pick L. Nature. 1997; 358: 552-555Crossref Scopus (161) Google Scholar), general transcription factors (8Um M. Li C. Manley J. Mol. Cell. Biol. 1995; 15: 5007-5016Crossref PubMed Scopus (86) Google Scholar, 9Zhang H. Caltron K.M. Abate-Shen C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1764-1769Crossref PubMed Scopus (131) Google Scholar, 10Zhu A. Kuziora M. J. Biol. Chem. 1997; 271: 20993-20996Abstract Full Text Full Text PDF Scopus (18) Google Scholar), and other types of proteins (11Agulnick A.D. Taria M. Breen J.J. Tanaka T. Bawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (292) Google Scholar, 12Kawabe T. Muslin A. Korsmeyer S.J. Nature. 1997; 385: 454-458Crossref PubMed Scopus (177) Google Scholar), have been identified to interact with homeodomain proteins. These protein/protein interactions modulate specific functions of homeodomain proteins, including their DNA binding affinities and specificities (3Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. Cell. 1994; 78: 603-615Abstract Full Text PDF PubMed Scopus (331) Google Scholar), transcription regulatory activities (13Lichtsteiner S. Tjian R. EMBO J. 1995; 14: 3937-3945Crossref PubMed Scopus (54) Google Scholar), and specific biological functions (11Agulnick A.D. Taria M. Breen J.J. Tanaka T. Bawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (292) Google Scholar, 12Kawabe T. Muslin A. Korsmeyer S.J. Nature. 1997; 385: 454-458Crossref PubMed Scopus (177) Google Scholar).14-3-3 proteins are highly related dimeric factors found in eukaryotic organisms, including yeast, Drosophila, plants, and mammals (14Aitken A. Trends Biochem. Sci. 1995; 20: 95-97Abstract Full Text PDF PubMed Scopus (264) Google Scholar). Members of the 14-3-3 family are involved in regulation of the enzymatic activities of tyrosine and tryptophan hydroxylases and protein kinase C, exocytosis, and the cell cycle (14Aitken A. Trends Biochem. Sci. 1995; 20: 95-97Abstract Full Text PDF PubMed Scopus (264) Google Scholar). Several important signaling proteins such as Raf-1, Bcr, phosphatidylinositol 3-kinase, and polyoma middle T antigen have been found to interact with 14-3-3 (15Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 16Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 17Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar, 18Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 19Pallas D.C. Fu H. Haehnel L.C. Weller W. Collier R.J. Roderts T.M. Science. 1994; 265: 535-537Crossref PubMed Scopus (148) Google Scholar, 20Reuther G.W. Fu H. Cripe L.D. Collier R.J. Penderpast A.M. Science. 1994; 266: 129-133Crossref PubMed Scopus (209) Google Scholar, 21Bonnefoy-Berard N. Liu Y.-C. von Villebrand M. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (133) Google Scholar, 22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 23Meller N. Liu Y.-C. Collins T.L. Bonnefoy-Berard N. Baier G. Isakov N. Altman A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar). A suggested role for 14-3-3 proteins is to function as scaffolds or adaptors to mediate interactions between different signaling proteins (24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar, 25Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (437) Google Scholar, 26Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar). It has been shown that 14-3-3 proteins can activate Raf-1 (15Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 27Li S. Janosch P. Tanji M. Rosenfeld G.C. Waymire J.C. Mischak H. Kolch W. Sedivy J.M. EMBO J. 1995; 14: 685-696Crossref PubMed Scopus (154) Google Scholar) and that they are required for specific Ras/mitogen-activated protein kinase signaling pathways in yeast and Drosophila (28Kockel L. Vorbruggen G. Jackle H. Mlodzik M. Bohmann D. Genes Dev. 1997; 11: 1140-1147Crossref PubMed Scopus (80) Google Scholar, 29Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (39) Google Scholar, 30Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar, 31Roberts R.L. Mosch H.-U. Fink G.R. Cell. 1997; 89: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).The Tlx-2 homeobox gene belongs to the HOX11family, in which three members, HOX11/Tlx-1, Tlx-2, and Tlx-3, have been identified (32Raju K. Tang S. Dubé I.D. Kamel-Reid S. Bryce D.M. Breitman M.L. Mech. Dev. 1993; 44: 51-64Crossref PubMed Scopus (70) Google Scholar, 33Dear T.N. Sanchez-Garcia I. Rabbitts T.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4431-4435Crossref PubMed Scopus (117) Google Scholar). The ectopic activation of HOX11/Tlx-1 in T cells by chromosomal translocations results in malignant transformation (34Dubé I.D. Kamel-Reid D. Yuan C.C. Lu M. Wu X. Corpus G. Raimond S.C. Crist W.M. Carroll A.J Minowada J. Baker J. Blood. 1991; 78: 2996-3003Crossref PubMed Google Scholar, 35Kennedy M.A. Gonzalez-Sarmiento R. Kees U.R. Lampert F. Dear N. Boehm T. Rabbitts T.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8900-8904Crossref PubMed Scopus (238) Google Scholar, 36Hatano M. Roberts C.W.M. Minden M. Crist W.M. Korsmeyers S.J. Science. 1991; 253: 79-82Crossref PubMed Scopus (371) Google Scholar, 37Lu M. Gong Z.Y. Shen W.F. Ho D.A. EMBO J. 1991; 10: 2905-2910Crossref PubMed Scopus (130) Google Scholar). Gene targeting experiments have shown that Tlx-1is required for the formation of mouse spleen (38Roberts C.W. Shutter J.R. Korsmeyer S.J. Nature. 1994; 368: 747-749Crossref PubMed Scopus (235) Google Scholar, 39Dear T.N. Colledge W.H. Carlton M.B. Lavenir I. Larson T. Smith A.J. Warren A.J. Evans M.J. Sofroniew M.V. Rabbitts T.H. Development (Camb.). 1995; 121: 2909-2915PubMed Google Scholar), whereasTlx-2 is required for mouse gastrulation and mesoderm formation. 1S. J. Tang, T.-C. Suen, R. R. McInnes, and M. Buchwald, unpublished results.1S. J. Tang, T.-C. Suen, R. R. McInnes, and M. Buchwald, unpublished results. During mouse embryogenesis, Tlx-2 is expressed in the primitive streak during gastrulation, in the neural ectoderm and the neural fold during neurulation, and later in the developing nervous system.1To obtain insight into the regulation of TLX-2 function, we searched for proteins that associate with TLX-2 in vivo. This paper describes an in vitro and in vivo interaction between TLX-2 and the mouse 14-3-3η protein. The biological relevance of this interaction has been established at multiple levels: 14-3-3η enhances the nuclear localization of TLX-2; Tlx-2 and 14-3-3η are coexpressed in the developing PNS2 and in PC12 cells; and TLX-2 and 14-3-3η cooperatively suppress neurite outgrowth of PC12 cells. Our findings provide the first evidence that a 14-3-3 protein can act as a cofactor for homeodomain transcription factors and suggest that the function of the TLX-2 homeodomain protein is modulated by specific signaling mediated by 14-3-3 proteins.DISCUSSIONIn vitro and in vivo interactions between the TLX-2 homeodomain and 14-3-3η proteins have been demonstrated in this study. The biological significance of the interaction is supported by several observations: 14-3-3η increases the nuclear localization of TLX-2; Tlx-2 and 14-3-3η are coexpressed in the developing PNS and in PC12 cells; and TLX-2 and 14-3-3η cooperatively suppress neurite outgrowth of PC12 cells induced by NGF. Recent studies have suggested that 14-3-3 proteins can bind to the phosphoserine in an RSXpSXP or RX(Y/F)XpSXP motif in various signaling proteins (46Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar, 47Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). However, such motifs are not present in the amino acid sequence of TLX-2, indicating that a different binding mechanism or motif mediates its interaction with 14-3-3η. Notably, these motifs are also not found in the peptide of platelet glycoprotein Ib-α that mediates its interaction with 14-3-3 (48Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and the two motifs (NRHpSLP and RLGpSTF) that are implicated in 14-3-3 binding of CbI share little homology with the above consensus binding sites, but can cooperatively confer stable binding to 14-3-3 (49Liu Y.-C. Liu Y. Elly C. Yoshida H. Lipkowitz S. Altman A. J. Biol. Chem. 1997; 272: 9979-9985Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Nonetheless, serine-rich regions are present in both the N- and C-terminal portions of TLX-2 (32Raju K. Tang S. Dubé I.D. Kamel-Reid S. Bryce D.M. Breitman M.L. Mech. Dev. 1993; 44: 51-64Crossref PubMed Scopus (70) Google Scholar). It will be interesting for further experiments to determine whether the interaction between TLX-2 and 14-3-3η proteins is also phosphoserine-dependentRecent studies have identified a variety of cofactors for homeodomain transcription factors; many of them are also involved in transcription regulation (see the Introduction). Although modulation of the DNA binding affinity and specificity of homeoproteins by their cofactors has been strongly suggested, the functional significance of their interactions is often unclear in a biological context. Our findings of the association of 14-3-3η with TLX-2 define a new class of cofactor for homeodomain transcription factors. Given the well established role of 14-3-3 proteins in signaling, our data provide a direct connection between the homeodomain transcription factors and cell signaling and suggest a novel mechanism for regulation of the function of homeodomain proteins during development.The biological functions of 14-3-3 proteins are poorly understood. Recent studies have suggested that, through interacting with Cdc25C, 14-3-3 regulates entry into the cell cycle (50Peng C.-Y. Graves P.R. Thomas R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1177) Google Scholar) and that, through interacting with BAD, 14-3-3 prevents apoptosis by releasing Bcl-XL (51Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). Our finding of the association of 14-3-3η with the TLX-2 homeodomain protein suggests a role of 14-3-3 proteins in regulation of developmental processes. Indeed, we observed a cooperative suppression of neurite outgrowth by these two factors (Fig. 5 B). The requirement of 14-3-3 proteins for specific developmental pathways has also been demonstrated recently in both yeast and Drosophila(28Kockel L. Vorbruggen G. Jackle H. Mlodzik M. Bohmann D. Genes Dev. 1997; 11: 1140-1147Crossref PubMed Scopus (80) Google Scholar, 29Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (39) Google Scholar, 30Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar, 31Roberts R.L. Mosch H.-U. Fink G.R. Cell. 1997; 89: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).14-3-3 proteins form homo- and heterodimers (44Jones D.H. Ley S. Aitken A. FEBS Lett. 1995; 368: 55-58Crossref PubMed Scopus (206) Google Scholar) in which the amino-terminal helices of the two subunits contact one another (25Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (437) Google Scholar,26Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar). Previous studies have suggested that such dimeric molecules may function as adaptors or scaffolds to mediate interactions between different proteins (22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar, 52Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). For instance, it has been shown that 14-3-3 proteins mediate the interaction between Raf and Bcr proteins and probably between Raf and Cdc25 and between Raf and A20 (22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar,52Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The association between 14-3-3η and TLX-2 suggests that the 14-3-3 protein may bridge an interaction between TLX-2 and another as yet uncharacterized protein. Given that 14-3-3 proteins are associated with numerous signaling kinases, it is likely that TLX-2 may interact with one or more of those kinases. Since phosphorylation and dephosphorylation serve as a major mechanism regulating the nuclear localization of transcription factors (53Vandromme M. Gauthier-Rouviere C. Lamb N. Fernandez A. Trends Biochem. Sci. 1996; 21: 59-64Abstract Full Text PDF PubMed Scopus (159) Google Scholar), the observed enhancement of TLX-2 nuclear localization by 14-3-3η may be attributed to a 14-3-3η-mediated interaction between TLX-2 and a signaling kinase that modulates the phosphorylation state of TLX-2. Taken together, our results suggest that one likely mechanism that controls the developmental function of TLX-2 is the regulation of its cellular distribution by signaling mediated by 14-3-3 proteins. Homeobox genes encode an evolutionarily conserved superfamily of transcription factors that play vital roles in various aspects of development (1Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1730) Google Scholar). A number of cofactors, including homeoproteins (2Xue D. Tu Y. Chalfie M. Science. 1993; 261: 1324-1328Crossref PubMed Scopus (181) Google Scholar, 3Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. Cell. 1994; 78: 603-615Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 4Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar), specific transcription factors (5Grueneberg D. Natesan S. Alexandre C. Gilman M. Science. 1992; 257: 1089-1095Crossref PubMed Scopus (256) Google Scholar, 6Guichet A. Copeland J.W.R. Erdelyi M. Hlousek D. Zavorszky P. Ho J. Brown S. Percival-Smith A. Krause H.M. Ephrussi A. Nature. 1997; 385: 548-552Crossref PubMed Scopus (158) Google Scholar, 7Yu Y. Li W. Yussa M. Han W. Perrimon N. Pick L. Nature. 1997; 358: 552-555Crossref Scopus (161) Google Scholar), general transcription factors (8Um M. Li C. Manley J. Mol. Cell. Biol. 1995; 15: 5007-5016Crossref PubMed Scopus (86) Google Scholar, 9Zhang H. Caltron K.M. Abate-Shen C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1764-1769Crossref PubMed Scopus (131) Google Scholar, 10Zhu A. Kuziora M. J. Biol. Chem. 1997; 271: 20993-20996Abstract Full Text Full Text PDF Scopus (18) Google Scholar), and other types of proteins (11Agulnick A.D. Taria M. Breen J.J. Tanaka T. Bawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (292) Google Scholar, 12Kawabe T. Muslin A. Korsmeyer S.J. Nature. 1997; 385: 454-458Crossref PubMed Scopus (177) Google Scholar), have been identified to interact with homeodomain proteins. These protein/protein interactions modulate specific functions of homeodomain proteins, including their DNA binding affinities and specificities (3Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. Cell. 1994; 78: 603-615Abstract Full Text PDF PubMed Scopus (331) Google Scholar), transcription regulatory activities (13Lichtsteiner S. Tjian R. EMBO J. 1995; 14: 3937-3945Crossref PubMed Scopus (54) Google Scholar), and specific biological functions (11Agulnick A.D. Taria M. Breen J.J. Tanaka T. Bawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (292) Google Scholar, 12Kawabe T. Muslin A. Korsmeyer S.J. Nature. 1997; 385: 454-458Crossref PubMed Scopus (177) Google Scholar). 14-3-3 proteins are highly related dimeric factors found in eukaryotic organisms, including yeast, Drosophila, plants, and mammals (14Aitken A. Trends Biochem. Sci. 1995; 20: 95-97Abstract Full Text PDF PubMed Scopus (264) Google Scholar). Members of the 14-3-3 family are involved in regulation of the enzymatic activities of tyrosine and tryptophan hydroxylases and protein kinase C, exocytosis, and the cell cycle (14Aitken A. Trends Biochem. Sci. 1995; 20: 95-97Abstract Full Text PDF PubMed Scopus (264) Google Scholar). Several important signaling proteins such as Raf-1, Bcr, phosphatidylinositol 3-kinase, and polyoma middle T antigen have been found to interact with 14-3-3 (15Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 16Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 17Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar, 18Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 19Pallas D.C. Fu H. Haehnel L.C. Weller W. Collier R.J. Roderts T.M. Science. 1994; 265: 535-537Crossref PubMed Scopus (148) Google Scholar, 20Reuther G.W. Fu H. Cripe L.D. Collier R.J. Penderpast A.M. Science. 1994; 266: 129-133Crossref PubMed Scopus (209) Google Scholar, 21Bonnefoy-Berard N. Liu Y.-C. von Villebrand M. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (133) Google Scholar, 22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 23Meller N. Liu Y.-C. Collins T.L. Bonnefoy-Berard N. Baier G. Isakov N. Altman A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar). A suggested role for 14-3-3 proteins is to function as scaffolds or adaptors to mediate interactions between different signaling proteins (24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar, 25Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (437) Google Scholar, 26Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar). It has been shown that 14-3-3 proteins can activate Raf-1 (15Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 27Li S. Janosch P. Tanji M. Rosenfeld G.C. Waymire J.C. Mischak H. Kolch W. Sedivy J.M. EMBO J. 1995; 14: 685-696Crossref PubMed Scopus (154) Google Scholar) and that they are required for specific Ras/mitogen-activated protein kinase signaling pathways in yeast and Drosophila (28Kockel L. Vorbruggen G. Jackle H. Mlodzik M. Bohmann D. Genes Dev. 1997; 11: 1140-1147Crossref PubMed Scopus (80) Google Scholar, 29Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (39) Google Scholar, 30Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar, 31Roberts R.L. Mosch H.-U. Fink G.R. Cell. 1997; 89: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The Tlx-2 homeobox gene belongs to the HOX11family, in which three members, HOX11/Tlx-1, Tlx-2, and Tlx-3, have been identified (32Raju K. Tang S. Dubé I.D. Kamel-Reid S. Bryce D.M. Breitman M.L. Mech. Dev. 1993; 44: 51-64Crossref PubMed Scopus (70) Google Scholar, 33Dear T.N. Sanchez-Garcia I. Rabbitts T.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4431-4435Crossref PubMed Scopus (117) Google Scholar). The ectopic activation of HOX11/Tlx-1 in T cells by chromosomal translocations results in malignant transformation (34Dubé I.D. Kamel-Reid D. Yuan C.C. Lu M. Wu X. Corpus G. Raimond S.C. Crist W.M. Carroll A.J Minowada J. Baker J. Blood. 1991; 78: 2996-3003Crossref PubMed Google Scholar, 35Kennedy M.A. Gonzalez-Sarmiento R. Kees U.R. Lampert F. Dear N. Boehm T. Rabbitts T.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8900-8904Crossref PubMed Scopus (238) Google Scholar, 36Hatano M. Roberts C.W.M. Minden M. Crist W.M. Korsmeyers S.J. Science. 1991; 253: 79-82Crossref PubMed Scopus (371) Google Scholar, 37Lu M. Gong Z.Y. Shen W.F. Ho D.A. EMBO J. 1991; 10: 2905-2910Crossref PubMed Scopus (130) Google Scholar). Gene targeting experiments have shown that Tlx-1is required for the formation of mouse spleen (38Roberts C.W. Shutter J.R. Korsmeyer S.J. Nature. 1994; 368: 747-749Crossref PubMed Scopus (235) Google Scholar, 39Dear T.N. Colledge W.H. Carlton M.B. Lavenir I. Larson T. Smith A.J. Warren A.J. Evans M.J. Sofroniew M.V. Rabbitts T.H. Development (Camb.). 1995; 121: 2909-2915PubMed Google Scholar), whereasTlx-2 is required for mouse gastrulation and mesoderm formation. 1S. J. Tang, T.-C. Suen, R. R. McInnes, and M. Buchwald, unpublished results.1S. J. Tang, T.-C. Suen, R. R. McInnes, and M. Buchwald, unpublished results. During mouse embryogenesis, Tlx-2 is expressed in the primitive streak during gastrulation, in the neural ectoderm and the neural fold during neurulation, and later in the developing nervous system.1To obtain insight into the regulation of TLX-2 function, we searched for proteins that associate with TLX-2 in vivo. This paper describes an in vitro and in vivo interaction between TLX-2 and the mouse 14-3-3η protein. The biological relevance of this interaction has been established at multiple levels: 14-3-3η enhances the nuclear localization of TLX-2; Tlx-2 and 14-3-3η are coexpressed in the developing PNS2 and in PC12 cells; and TLX-2 and 14-3-3η cooperatively suppress neurite outgrowth of PC12 cells. Our findings provide the first evidence that a 14-3-3 protein can act as a cofactor for homeodomain transcription factors and suggest that the function of the TLX-2 homeodomain protein is modulated by specific signaling mediated by 14-3-3 proteins. DISCUSSIONIn vitro and in vivo interactions between the TLX-2 homeodomain and 14-3-3η proteins have been demonstrated in this study. The biological significance of the interaction is supported by several observations: 14-3-3η increases the nuclear localization of TLX-2; Tlx-2 and 14-3-3η are coexpressed in the developing PNS and in PC12 cells; and TLX-2 and 14-3-3η cooperatively suppress neurite outgrowth of PC12 cells induced by NGF. Recent studies have suggested that 14-3-3 proteins can bind to the phosphoserine in an RSXpSXP or RX(Y/F)XpSXP motif in various signaling proteins (46Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar, 47Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). However, such motifs are not present in the amino acid sequence of TLX-2, indicating that a different binding mechanism or motif mediates its interaction with 14-3-3η. Notably, these motifs are also not found in the peptide of platelet glycoprotein Ib-α that mediates its interaction with 14-3-3 (48Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and the two motifs (NRHpSLP and RLGpSTF) that are implicated in 14-3-3 binding of CbI share little homology with the above consensus binding sites, but can cooperatively confer stable binding to 14-3-3 (49Liu Y.-C. Liu Y. Elly C. Yoshida H. Lipkowitz S. Altman A. J. Biol. Chem. 1997; 272: 9979-9985Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Nonetheless, serine-rich regions are present in both the N- and C-terminal portions of TLX-2 (32Raju K. Tang S. Dubé I.D. Kamel-Reid S. Bryce D.M. Breitman M.L. Mech. Dev. 1993; 44: 51-64Crossref PubMed Scopus (70) Google Scholar). It will be interesting for further experiments to determine whether the interaction between TLX-2 and 14-3-3η proteins is also phosphoserine-dependentRecent studies have identified a variety of cofactors for homeodomain transcription factors; many of them are also involved in transcription regulation (see the Introduction). Although modulation of the DNA binding affinity and specificity of homeoproteins by their cofactors has been strongly suggested, the functional significance of their interactions is often unclear in a biological context. Our findings of the association of 14-3-3η with TLX-2 define a new class of cofactor for homeodomain transcription factors. Given the well established role of 14-3-3 proteins in signaling, our data provide a direct connection between the homeodomain transcription factors and cell signaling and suggest a novel mechanism for regulation of the function of homeodomain proteins during development.The biological functions of 14-3-3 proteins are poorly understood. Recent studies have suggested that, through interacting with Cdc25C, 14-3-3 regulates entry into the cell cycle (50Peng C.-Y. Graves P.R. Thomas R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1177) Google Scholar) and that, through interacting with BAD, 14-3-3 prevents apoptosis by releasing Bcl-XL (51Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). Our finding of the association of 14-3-3η with the TLX-2 homeodomain protein suggests a role of 14-3-3 proteins in regulation of developmental processes. Indeed, we observed a cooperative suppression of neurite outgrowth by these two factors (Fig. 5 B). The requirement of 14-3-3 proteins for specific developmental pathways has also been demonstrated recently in both yeast and Drosophila(28Kockel L. Vorbruggen G. Jackle H. Mlodzik M. Bohmann D. Genes Dev. 1997; 11: 1140-1147Crossref PubMed Scopus (80) Google Scholar, 29Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (39) Google Scholar, 30Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar, 31Roberts R.L. Mosch H.-U. Fink G.R. Cell. 1997; 89: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).14-3-3 proteins form homo- and heterodimers (44Jones D.H. Ley S. Aitken A. FEBS Lett. 1995; 368: 55-58Crossref PubMed Scopus (206) Google Scholar) in which the amino-terminal helices of the two subunits contact one another (25Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (437) Google Scholar,26Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar). Previous studies have suggested that such dimeric molecules may function as adaptors or scaffolds to mediate interactions between different proteins (22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar, 52Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). For instance, it has been shown that 14-3-3 proteins mediate the interaction between Raf and Bcr proteins and probably between Raf and Cdc25 and between Raf and A20 (22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar,52Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The association between 14-3-3η and TLX-2 suggests that the 14-3-3 protein may bridge an interaction between TLX-2 and another as yet uncharacterized protein. Given that 14-3-3 proteins are associated with numerous signaling kinases, it is likely that TLX-2 may interact with one or more of those kinases. Since phosphorylation and dephosphorylation serve as a major mechanism regulating the nuclear localization of transcription factors (53Vandromme M. Gauthier-Rouviere C. Lamb N. Fernandez A. Trends Biochem. Sci. 1996; 21: 59-64Abstract Full Text PDF PubMed Scopus (159) Google Scholar), the observed enhancement of TLX-2 nuclear localization by 14-3-3η may be attributed to a 14-3-3η-mediated interaction between TLX-2 and a signaling kinase that modulates the phosphorylation state of TLX-2. Taken together, our results suggest that one likely mechanism that controls the developmental function of TLX-2 is the regulation of its cellular distribution by signaling mediated by 14-3-3 proteins. In vitro and in vivo interactions between the TLX-2 homeodomain and 14-3-3η proteins have been demonstrated in this study. The biological significance of the interaction is supported by several observations: 14-3-3η increases the nuclear localization of TLX-2; Tlx-2 and 14-3-3η are coexpressed in the developing PNS and in PC12 cells; and TLX-2 and 14-3-3η cooperatively suppress neurite outgrowth of PC12 cells induced by NGF. Recent studies have suggested that 14-3-3 proteins can bind to the phosphoserine in an RSXpSXP or RX(Y/F)XpSXP motif in various signaling proteins (46Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar, 47Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). However, such motifs are not present in the amino acid sequence of TLX-2, indicating that a different binding mechanism or motif mediates its interaction with 14-3-3η. Notably, these motifs are also not found in the peptide of platelet glycoprotein Ib-α that mediates its interaction with 14-3-3 (48Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and the two motifs (NRHpSLP and RLGpSTF) that are implicated in 14-3-3 binding of CbI share little homology with the above consensus binding sites, but can cooperatively confer stable binding to 14-3-3 (49Liu Y.-C. Liu Y. Elly C. Yoshida H. Lipkowitz S. Altman A. J. Biol. Chem. 1997; 272: 9979-9985Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Nonetheless, serine-rich regions are present in both the N- and C-terminal portions of TLX-2 (32Raju K. Tang S. Dubé I.D. Kamel-Reid S. Bryce D.M. Breitman M.L. Mech. Dev. 1993; 44: 51-64Crossref PubMed Scopus (70) Google Scholar). It will be interesting for further experiments to determine whether the interaction between TLX-2 and 14-3-3η proteins is also phosphoserine-dependent Recent studies have identified a variety of cofactors for homeodomain transcription factors; many of them are also involved in transcription regulation (see the Introduction). Although modulation of the DNA binding affinity and specificity of homeoproteins by their cofactors has been strongly suggested, the functional significance of their interactions is often unclear in a biological context. Our findings of the association of 14-3-3η with TLX-2 define a new class of cofactor for homeodomain transcription factors. Given the well established role of 14-3-3 proteins in signaling, our data provide a direct connection between the homeodomain transcription factors and cell signaling and suggest a novel mechanism for regulation of the function of homeodomain proteins during development. The biological functions of 14-3-3 proteins are poorly understood. Recent studies have suggested that, through interacting with Cdc25C, 14-3-3 regulates entry into the cell cycle (50Peng C.-Y. Graves P.R. Thomas R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1177) Google Scholar) and that, through interacting with BAD, 14-3-3 prevents apoptosis by releasing Bcl-XL (51Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). Our finding of the association of 14-3-3η with the TLX-2 homeodomain protein suggests a role of 14-3-3 proteins in regulation of developmental processes. Indeed, we observed a cooperative suppression of neurite outgrowth by these two factors (Fig. 5 B). The requirement of 14-3-3 proteins for specific developmental pathways has also been demonstrated recently in both yeast and Drosophila(28Kockel L. Vorbruggen G. Jackle H. Mlodzik M. Bohmann D. Genes Dev. 1997; 11: 1140-1147Crossref PubMed Scopus (80) Google Scholar, 29Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (39) Google Scholar, 30Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar, 31Roberts R.L. Mosch H.-U. Fink G.R. Cell. 1997; 89: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). 14-3-3 proteins form homo- and heterodimers (44Jones D.H. Ley S. Aitken A. FEBS Lett. 1995; 368: 55-58Crossref PubMed Scopus (206) Google Scholar) in which the amino-terminal helices of the two subunits contact one another (25Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (437) Google Scholar,26Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar). Previous studies have suggested that such dimeric molecules may function as adaptors or scaffolds to mediate interactions between different proteins (22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar, 52Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). For instance, it has been shown that 14-3-3 proteins mediate the interaction between Raf and Bcr proteins and probably between Raf and Cdc25 and between Raf and A20 (22Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 24Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar,52Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The association between 14-3-3η and TLX-2 suggests that the 14-3-3 protein may bridge an interaction between TLX-2 and another as yet uncharacterized protein. Given that 14-3-3 proteins are associated with numerous signaling kinases, it is likely that TLX-2 may interact with one or more of those kinases. Since phosphorylation and dephosphorylation serve as a major mechanism regulating the nuclear localization of transcription factors (53Vandromme M. Gauthier-Rouviere C. Lamb N. Fernandez A. Trends Biochem. Sci. 1996; 21: 59-64Abstract Full Text PDF PubMed Scopus (159) Google Scholar), the observed enhancement of TLX-2 nuclear localization by 14-3-3η may be attributed to a 14-3-3η-mediated interaction between TLX-2 and a signaling kinase that modulates the phosphorylation state of TLX-2. Taken together, our results suggest that one likely mechanism that controls the developmental function of TLX-2 is the regulation of its cellular distribution by signaling mediated by 14-3-3 proteins. We are grateful to Drs. J. Lightfoot, C. C. Hui, and M. Crackower for critical reading of the manuscript.