Abl Interactor 1 Promotes Tyrosine 296 Phosphorylation of Mammalian Enabled (Mena) by c-Abl Kinase

Abstract

Mammalian Enabled (Mena) is a mammalian homologue of Drosophila Enabled (Ena), which genetically interacts with Drosophila Abl tyrosine kinase. The signaling pathway involving c-Abl and Mena (Ena) is not fully understood. To find molecules that participate in the c-Abl/Mena pathway, we searched for Mena-binding proteins using a yeast two-hybrid system. We identified Abl interactor 1 (Abi-1), which is known to interact with c-Abl, as a binding protein for Mena. Binding analysis revealed that the Ena/Vasp homology 1 domain of Mena and the polyproline structure of Abi-1 are necessary for the interaction. The interaction between Mena and Abi-1 was also observed in a mammalian expression system. Importantly, Abi-1 dramatically promoted c-Abl-mediated tyrosine phosphorylation of Mena but not other substrates such as c-Cbl. Mutational analysis demonstrated that the phosphorylation site of Mena is Tyr-296. Our results suggest that Abi-1 regulates c-Abl-mediated phosphorylation of Mena by interacting with both proteins. Mammalian Enabled (Mena) is a mammalian homologue of Drosophila Enabled (Ena), which genetically interacts with Drosophila Abl tyrosine kinase. The signaling pathway involving c-Abl and Mena (Ena) is not fully understood. To find molecules that participate in the c-Abl/Mena pathway, we searched for Mena-binding proteins using a yeast two-hybrid system. We identified Abl interactor 1 (Abi-1), which is known to interact with c-Abl, as a binding protein for Mena. Binding analysis revealed that the Ena/Vasp homology 1 domain of Mena and the polyproline structure of Abi-1 are necessary for the interaction. The interaction between Mena and Abi-1 was also observed in a mammalian expression system. Importantly, Abi-1 dramatically promoted c-Abl-mediated tyrosine phosphorylation of Mena but not other substrates such as c-Cbl. Mutational analysis demonstrated that the phosphorylation site of Mena is Tyr-296. Our results suggest that Abi-1 regulates c-Abl-mediated phosphorylation of Mena by interacting with both proteins. c-abl was first identified as a protooncogene of v-abl carried by the Abelson murine leukemia virus, which causes oncogenic transformation of immature B lymphocytes (1Goff S.P. D'Eustachio P. Ruddle F.H. Baltimore D. Science. 1982; 218: 1317-1319Crossref PubMed Scopus (36) Google Scholar, 2Wang J.Y. Ledley F. Goff S. Lee R. Groner Y. Baltimore D. Cell. 1984; 36: 349-356Abstract Full Text PDF PubMed Scopus (122) Google Scholar). The c-abl gene encodes a nonreceptor tyrosine kinase, c-Abl. c-Abl has been implicated in a variety of cellular processes including cell growth, reorganization of cytoskeleton, cell death, and stress responses (reviewed in Refs. 3Van Etten R.A. Trends Cell Biol. 1999; 9: 179-186Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 4Lanier L.M. Gertler F.B. Curr. Opin. Neurobiol. 2000; 10: 80-87Crossref PubMed Scopus (177) Google Scholar, 5Wang J.Y. Oncogene. 2000; 19: 5643-5650Crossref PubMed Scopus (250) Google Scholar). Oncogenic forms of Abl possess increased and unregulated kinase activity (6Ponticelli A.S. Whitlock C.A. Rosenberg N. Witte O.N. Cell. 1982; 29: 953-960Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 7Konopka J.B. Watanabe S.M. Witte O.N. Cell. 1984; 37: 1035-1042Abstract Full Text PDF PubMed Scopus (678) Google Scholar). This increased kinase activity is believed to activate signal transduction pathways, which ultimately leads to uncontrolled cell growth. c-Abl kinase is known to be regulated through several mechanisms. Trans-acting inhibitors and activators can regulate c-Abl kinase (8Pendergast A.M. Muller A.J. Havlik M.H. Clark R. McCormick F. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5927-5931Crossref PubMed Scopus (123) Google Scholar, 9Mayer B.J. Baltimore D. Mol. Cell. Biol. 1994; 14: 2883-2894Crossref PubMed Scopus (158) Google Scholar, 10Shishido T. Akagi T. Ouchi T. Georgescu M.M. Langdon W.Y. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6439-6444Crossref PubMed Scopus (24) Google Scholar, 11Shishido T. Akagi T. Chalmers A. Maeda M. Terada T. Georgescu M.M. Hanafusa H. Genes Cells. 2001; 6: 431-440Crossref PubMed Scopus (50) Google Scholar, 12Smith J.M. Mayer B.J. Front. Biosci. 2002; 7: d31-d42Crossref PubMed Google Scholar). In addition, c-Abl kinase can be intramolecularly folded, resulting in suppression of its activity (13Pluk H. Dorey K. Superti-Furga G. Cell. 2002; 108: 247-259Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Abl interactor 1 (Abi-1) was discovered as a c-Abl-binding protein that can inhibit v-Abl-mediated transformation of cultured cells (14Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (216) Google Scholar). Abi-1 contains an Src homology 3 (SH3) 1The abbreviations used are: SH3, Src homology 3; EVH1 and -2, Ena/Vasp homology 1 and 2, respectively; GST, glutathione S-transferase; aa, amino acids; HA, hemagglutinin.1The abbreviations used are: SH3, Src homology 3; EVH1 and -2, Ena/Vasp homology 1 and 2, respectively; GST, glutathione S-transferase; aa, amino acids; HA, hemagglutinin. domain and a polyproline structure. Both domains are involved in the interaction with c-Abl (14Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (216) Google Scholar). Abl interactor proteins exist as multiple forms in mammalian cells. E3B1 (15Biesova Z. Piccoli C. Wong W.T. Oncogene. 1997; 14: 233-241Crossref PubMed Scopus (90) Google Scholar) seems to be a splice variant of Abi-1, and Abi-2 (16Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar) is highly related to Abi-1 but encoded by a different gene. It has been reported that E3B1 (Abi-1) is involved in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation (17Scita G. Nordstrom J. Carbone R. Tenca P. Giardina G. Gutkind S. Bjarnegard M. Betsholtz C. Di Fiore P.P. Nature. 1999; 401: 290-293Crossref PubMed Scopus (283) Google Scholar). To understand the signal transduction pathway involving c-Abl, Drosophila genetics was applied to obtain genetically interacting molecules (18Gertler F.B. Doctor J.S. Hoffmann F.M. Science. 1990; 248: 857-860Crossref PubMed Scopus (118) Google Scholar, 19Gertler F.B. Comer A.R. Juang J.L. Ahern S.M. Clark M.J. Liebl E.C. Hoffmann F.M. Genes Dev. 1995; 9: 521-533Crossref PubMed Scopus (222) Google Scholar). Abl mutant flies die at the end of pupation or as young adults (20Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). Drosophila enabled (ena) was identified as a dosage-sensitive suppressor of mutations in Drosophila Abl (19Gertler F.B. Comer A.R. Juang J.L. Ahern S.M. Clark M.J. Liebl E.C. Hoffmann F.M. Genes Dev. 1995; 9: 521-533Crossref PubMed Scopus (222) Google Scholar). Mammalian enabled (mena) has been identified, and its function has been extensively investigated (20Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 21Lanier L.M. Gates M.A. Witke W. Menzies A.S. Wehman A.M. Macklis J.D. Kwiatkowski D. Soriano P. Gertler F.B. Neuron. 1999; 22: 313-325Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 22Bear J.E. Loureiro J.J. Libova I. Fassler R. Wehland J. Gertler F.B. Cell. 2000; 101: 717-728Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Mena contains an Ena/Vasp homology 1 (EVH1) domain, Arg/Leu/Glu-rich region, proline-rich region, and EVH2 domain (20Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). The EVH1 domain binds to proteins containing consensus sequence (D/E)FPPPPX(D/E) (23Prehoda K.E. Lee D.J. Lim W.A. Cell. 1999; 97: 471-480Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The EVH1 ligand motif is found in several proteins including a focal adhesion protein, Zyxin (24Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and ActA (25Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (332) Google Scholar). The proline-rich region of Mena is able to interact with profilin (20Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 26Ahern-Djamali S.M. Bachmann C. Hua P. Reddy S.K. Kastenmeier A.S. Walter U. Hoffmann F.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4977-4982Crossref PubMed Scopus (40) Google Scholar), and the EVH2 domain has regions implicated in G- and F-actin binding. In cultured fibroblasts, Mena is localized to focal contacts and the leading edge. Mena is implicated in cell motility through regulation of the actin cytoskeleton (22Bear J.E. Loureiro J.J. Libova I. Fassler R. Wehland J. Gertler F.B. Cell. 2000; 101: 717-728Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Although the interactions of Mena with actin and actin-binding proteins have been extensively characterized, the signaling pathways involving Mena have not been fully understood. With the hope of finding proteins that regulate the function of Mena, we performed yeast two-hybrid screening. We identified Abi-1 as a Mena-binding protein. Phosphorylation of Mena by c-Abl was greatly increased in the presence of Abi-1 in cultured cells. Tyr-296 of Mena was identified as a single phosphorylation site for c-Abl kinase. Our results suggest the functional importance of the linkage of these three molecules in signal transduction. cDNA Cloning and Expression Analysis—cDNAs for Mena and Mena(S) were amplified by PCR using a mouse spleen cDNA library (Clontech) as a template. An N-terminal primer (5′-AGATCTACCATGAGTGAACAGAGTATCTGT) and a reverse primer (5′-GAGGTGCAGGTGGTCCCAAGA) were used to amplify the 5′ halves. A forward primer (5′-TCTTGGGACCACCTGCACCTC) and a C-terminal reverse primer (5′-AGATCTTTATGCAGTGTTCGACTTGCTCAG) were used for the 3′ halves. The PCR products were subcloned into pCRBlunt (Invitrogen), and then their sequences were determined. Full-length cDNAs for Mena and Mena(S) were prepared by PCR using the amplified 5′ and 3′ fragments as templates. To examine the expression of mRNAs for Mena and Mena(S) by PCR, a forward primer (5′-TCTTGGGACCACCTGCACCTC) and a reverse primer (5′-CCAGCAGGGCACTCATTTCTTCC) were used. These primers were designed so as to encompass the region missing in Mena(S), so that the PCR products derived from cDNAs for Mena and Mena(S) could be distinguished. cDNAs derived from mouse 17-day embryos and mouse brains were purchased from Clontech. Antibodies—The cDNA fragment encoding the N-terminal region (amino acids (aa) 1–284) of Mena was inserted into expression vector pGEX-4T-1 (Amersham Biosciences). The glutathione S-transferase (GST)-Mena fusion protein was expressed in Escherichia coli cells and purified according to the standard method. Rabbits were immunized with the purified protein (MBL Inc.). The sera were first depleted of anti-GST antibodies by chromatography on a GST column, and then the anti-Mena was using The cDNA fragment encoding the Abi-1 was inserted into The fusion protein was purified as The purified protein was with and then into cells antibodies Abi-1 were according to the standard The antibodies were a from anti-GST and and cDNA of Mena(S) was subcloned into yeast expression vector The was into yeast and then two-hybrid was carried according to the using a domain fusion library in cDNA that in were and their activity was on The were of the encoded which to We to as Abi-1 in To examine the cDNA fragment encoding the Mena or of its was inserted into The cDNA fragment encoding the Abi-1 or of its was inserted into with were by PCR and by was with and and for activity were performed according to the Mammalian Expression cDNAs for Mena and Mena(S) were subcloned into mammalian expression The and were used to Mena and Mena(S) in cells. of Abi-1 were by PCR and by To the mutant and in which the tyrosine of Mena were with was carried with a according to the were by Mammalian expression from and were used to proteins with an N-terminal GST and Expression c-Abl and and T. Akagi T. Ouchi T. Georgescu M.M. Langdon W.Y. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6439-6444Crossref PubMed Scopus (24) Google Scholar) were from Baltimore and cells were in with and and cells from were cultured in with and from a mouse cell from H. F. J. PubMed Scopus Google Scholar). and were prepared from and The of were by which possess a in the c-abl Cell. 1991; Full Text PDF PubMed Scopus Google Scholar) from and The were prepared from embryos and according to the H. J. Cell Biol. PubMed Scopus Google Scholar). These were used for and The were cultured in with and In Binding the expression of fusion cells on were with 1 of expression using the cells were in of 1 1 and The was in a for at to The was with (Amersham for with at and then three with the The with the were from the by in The were by and with antibodies using (Amersham Biosciences). Phosphorylation cells were with 1 of expression and then in the containing inhibitors and The of were with or of the phosphorylation of the was with and then at for with The were with the containing and the with the were by with of the phosphorylation of was to the by for 3 at were then to the by at for with The were and then as was performed as K. H. T. J. H. M. M. K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). cells on were with by with antibodies and or was performed with an Cloning and of an of obtain we performed PCR using a mouse spleen cDNA library as a template. The 5′ and 3′ regions of cDNA were of the 3′ region of PCR a single was for the 5′ region not analysis of the PCR products for the 3′ region revealed that to reported mouse and the other to Full-length cDNAs for and its as were prepared by PCR using the amplified 5′ and 3′ To or not is we performed PCR analysis using cDNAs from mouse 17-day mouse and mouse as templates. To the PCR products for and primers were designed to encompass the region to of Mena. The PCR products for and were observed in cDNAs The expression of was in mouse spleen To examine the expression of Mena and Mena(S) at the protein we a expressed Mena. of of mouse B cell and and cells were by with the anti-Mena In addition, of cells with an expression encoding Mena or Mena(S) were also In with results (20Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar), protein to and were for its expression in the to a of Mena, the other the is Mena, was expressed in of cells and the on as expressed Mena in cells. that the for the of cells with the Mena expression was that for the of In to the protein, an protein was in and the protein the on as expressed Mena(S), the protein is These results suggest that Mena(S) is expressed in cell which is with the that the is expressed in of Mena-binding a molecules that participate in the c-Abl/Mena pathway in mammalian we searched for Mena-binding proteins using a yeast two-hybrid system. We performed with Mena(S) as the We that with Mena(S) to obtain binding Mena(S) of Mena, which contains the We used a cDNA library Mena(S) to be expressed in cell A and In were and were The results of sequence analysis of the are in I. to be which has been reported to to Mena (24Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). encoded a protein. of protein be encoded Abi-1, and encoded its Abi-1 was identified as a c-Abl-binding protein (14Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (216) Google Scholar). These results the that Abi-1 interacts with of the two-hybrid in a The EVH1 of Mena in the with the interaction between Mena and Abi-1 using the yeast two-hybrid system. we or not Mena as as Mena(S) binds to in the of 2, both Mena and Mena(S) to To the binding region for Abi-1 on Mena, we of Mena. We Mena into the EVH1 domain Arg/Leu/Glu-rich region proline-rich region and EVH2 domain The interactions of Abi-1 with the Mena fragments were in the EVH1 domain to A mutant the EVH1 domain not to we the region of Abi-1 that is involved in the binding to Mena. Abi-1 contains an domain and a polyproline structure the We C-terminal of Abi-1 and were Abi-1 was a mutant both the domain and the polyproline structure. was a mutant the polyproline structure. The interactions of these with Mena were in the of 2, the domain Abi-1 and with Mena. the other Abi-1 and not interact with Mena. We these results by with as an The Abi-1 and of interaction with Mena not These results that the polyproline but not the domain, of Abi-1 is for the interaction with Mena. Binding of Mena to Abi-1 in Mammalian interaction between Mena and Abi-1 was using a mammalian cell system. GST or was with Mena in cells. GST or was with glutathione and then the Mena was in Mena was with but not with and Abi-1 were in and were to in Abi-1 was with These results that expressed Mena and Abi-1 interact with other in cells. To interacts with was expressed in and then of proteins with was in Mena and c-Abl were with which is known to be a of c-Abl T. Akagi T. Ouchi T. Georgescu M.M. Langdon W.Y. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6439-6444Crossref PubMed Scopus (24) Google Scholar), was not nonreceptor tyrosine kinase, was not with these results suggest a interaction between Mena and Abi-1 in mammalian cells. Abi-1 c-Abl-mediated Phosphorylation of is reported to be a of Drosophila Abl (19Gertler F.B. Comer A.R. Juang J.L. Ahern S.M. Clark M.J. Liebl E.C. Hoffmann F.M. Genes Dev. 1995; 9: 521-533Crossref PubMed Scopus (222) Google Scholar). The finding that Mena interacts with Abi-1 to examine or not Abi-1 the phosphorylation of Mena by c-Abl cells were with expression for c-Abl, Abi-1, and was from the cell and its phosphorylation was investigated by with Phosphorylation of was was with c-Abl A in the phosphorylation of was observed was with Abi-1 in to c-Abl A mutant of c-Abl, not in the presence of Abi-1 that the kinase activity of c-Abl is for the phosphorylation of of the used for the of the phosphorylation of with an anti-GST that of in These results that Abi-1 promoted c-Abl-mediated phosphorylation of To the of we used as a for c-Abl T. Akagi T. Ouchi T. Georgescu M.M. Langdon W.Y. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6439-6444Crossref PubMed Scopus (24) Google Scholar). Expression encoding c-Abl, Abi-1, and were into cells. was from the cell with and then its phosphorylation was The of phosphorylation of by c-Abl not of the presence or of Abi-1 3 and We the phosphorylation of using the system. Phosphorylation of by c-Abl was not promoted in the presence of Abi-1 not These results the that Abi-1 the phosphorylation of Mena by c-Abl kinase. of Abi-1 on Phosphorylation of we to the Abi-1 domain involved in the of phosphorylation of Mena by c-Abl kinase. Phosphorylation analysis was performed with the of Abi-1 used for the in In addition, we in which a in the domain was by a of the expression encoding Abi-1 was with expression encoding c-Abl and into cells. was and with the The on the were with The of phosphorylation of in the of cells with Abi-1 was that with the Abi-1 A phosphorylation was observed and were expressed and the other Abi-1 not the phosphorylation of by c-Abl These results that both the domain and the polyproline structure of Abi-1 to the of phosphorylation of Mena by c-Abl kinase. Tyr-296 of Mena the Phosphorylation contains tyrosine and To which tyrosine in Mena is by c-Abl kinase, we by encoding c-Abl, Abi-1, and were into cells. were and their phosphorylation were by with The and and were at the the other the mutant was not that Tyr-296 was the site of The mutant in which tyrosine and were by was at the as the Mena, the mutant in which of the tyrosine were was not These results that Tyr-296 of Mena is the phosphorylation site in Mena for c-Abl kinase. The results of these mutant are in Abi-1 with Mena at the is localized to focal and the leading in (20Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). Although Abi-2 has been reported to be localized to in murine cells T. K. P. Pendergast A.M. Curr. Biol. 2001; Full Text Full Text PDF PubMed Scopus (118) Google Scholar), its has not been with that of Mena. We a Abi-1, and then the of Abi-1 and Mena was by in the that we Abi-1 in of mouse and and cells as as The observed for the of the phosphorylation of Abi-1 is known to be by Abl kinase A. G. Goff S.P. Oncogene. 2001; PubMed Scopus Google Scholar). These were observed as not The of Mena and Abi-1 in mouse was by Mena is localized at focal contacts and the leading Abi-1 is localized at the leading but not focal A the of the proteins at the leading edge. we or not their was by of c-Abl. We the of Abi-1 and Mena in in the of was observed in the of the proteins. This that the presence of c-Abl not the of protein. the of Mena and Abi-1 at the leading in the is with the finding that Abi-1 interacts with Mena and suggest functional between these molecules for the regulation of the Abi-1 was first discovered as an protein that can v-Abl-mediated transformation of cultured cells (14Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (216) Google Scholar). In the we have demonstrated that Mena also interacts with The EVH1 domain of Mena and the polyproline structure of Abi-1 are involved in the interaction. Our results suggest a linkage between c-Abl, Abi-1, and Mena. the phosphorylation of Mena by c-Abl was dramatically by The that the phosphorylation of and by c-Abl is not by Abi-1 the that Abi-1 c-Abl kinase activity for The results are not in with the finding that Drosophila and Abi-2 the phosphorylation of Drosophila Enabled by Abl J.L. Hoffmann F.M. Oncogene. 1999; PubMed Scopus Google Scholar) but also a for the The interaction between c-Abl, Abi-1, and Mena the presence of a of the three proteins. We to using a yeast system. such was not for a was not observed is that the binding site for c-Abl on Abi-1 is by Mena. Our binding revealed that the polyproline structure of Abi-1 is for the binding to Mena. the other the domain of Abi-1 is for the binding to c-Abl, and the polyproline structure the binding (14Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (216) Google Scholar). c-Abl to Abi-1 through the interaction with the domain the polyproline structure is by Mena. These interactions not be for the of a but are for the of the phosphorylation of Mena by c-Abl. It is that the phosphorylation of Mena by c-Abl is promoted to in the presence of Abi-1 the domain or the polyproline structure. for is that Abi-1 in c-Abl Mena binding to these proteins. The binding of Abi-1 to c-Abl c-Abl with for the Mena but not other The binding of Abi-1 to Mena also the of the phosphorylation site of Mena. In Abi-1 is not a for Mena and c-Abl but a that the of Mena to c-Abl by binding to both a and kinase. It is to that the phosphorylation of Tyr-296 regulates the function of Mena. that Tyr-296 is in the of the proline-rich region, the phosphorylation the interaction of Mena with proteins that to region, such as Phosphorylation of Tyr-296 also a in Mena, which the of domains and from the phosphorylation The of Mena(S) is not at Mena(S) the motif that is in the proline-rich region of Mena. Mena(S) is expressed in that its function be to It has been reported that the proline-rich region of Mena is for cell motility but for J.J. J.E. Kwiatkowski Gertler F.B. Mol. Biol. Cell. 2002; PubMed Scopus Google Scholar). Mena(S) regulate reorganization of the actin cytoskeleton in Mena(S) is not able to to pathways by with the interaction of Mena with Mena-binding proteins. It is that the region in Mena(S) is to the phosphorylation site of Mena. Phosphorylation have different on Mena and Mena(S) In we have that Abi-1 binds to Mena and c-Abl and the phosphorylation of Mena by c-Abl. Our results to the of the interaction of the three c-Abl, Abi-1, and Mena, a signaling pathway of c-Abl kinase. We and Terada for We are to and for We are to Goff and for We are to for and of the