From the
School of Life Science, Tokyo
University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392,
Japan, ||Osaka Bioscience Institute, 6-2-4
Furuedai, Osaka 565-0874, Japan, the **Department of Molecular Oncology, Osaka
University Graduate School of Medicine, 2-2, Yamada-oka Suita, Osaka
565-0871, Japan,
Japan Science
and Technology Corporation, Saitama 332-0012, Japan, and
Japan Society for the Promotion of
Science, Tokyo 102-8471, Japan
Received for publication, February 10, 2003 , and in revised form, March 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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Abl interactor 1 (Abi-1) was discovered as a c-Abl-binding protein that can inhibit v-Abl-mediated transformation of cultured cells (14). Abi-1 contains an Src homology 3 (SH3)1 domain and a polyproline structure. Both domains are involved in the interaction with c-Abl (14). Abl interactor proteins exist as multiple forms in mammalian cells. E3B1 (15) seems to be a splice variant of Abi-1, and Abi-2 (16) 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 (17).
To understand the signal transduction pathway involving c-Abl, Drosophila genetics was applied to obtain genetically interacting molecules (18, 19). Abl mutant flies die at the end of pupation or as young adults (20). Drosophila enabled (ena) was identified as a dosage-sensitive suppressor of mutations in Drosophila Abl (19). Mammalian enabled (mena) has been identified, and its function has been extensively investigated (2022). Mena contains an Ena/Vasp homology 1 (EVH1) domain, Arg/Leu/Glu-rich region, proline-rich region, and EVH2 domain (20). The EVH1 domain binds to proteins containing consensus sequence (D/E)FPPPPX(D/E) (23). The EVH1 ligand motif is found in several proteins including a focal adhesion protein, Zyxin (24), and ActA (25). The proline-rich region of Mena is able to interact with profilin (20, 26), 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 (22).
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.
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EXPERIMENTAL PROCEDURES |
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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.
AntibodiesThe cDNA fragment encoding the N-terminal region (amino acids (aa) 1284) 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 antibody was affinity-purified using antigen-coupled beads.
The cDNA fragment encoding the full-length Abi-1 was inserted into pGEX-4T-1. The GST-Abi-1 fusion protein was purified as described above. The purified protein was mixed with Immun EasyTM Mouse Adjuvant (Qiagen) and then injected into BALB/c mice. Hybridoma cells producing antibodies against Abi-1 were obtained according to the standard protocol.
The following antibodies were used: anti-Abl (8E9; Pharmingen), anti-c-Cbl (C15; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-c-Src (327; a gift from Joan Brugge), anti-HA (Y11; Santa Cruz Biotechnology), anti-FLAG (M2; Sigma), anti-GST (Santa Cruz Biotechnology), and anti-phosphotyrosine (4G10; Upstate Biotechnology, Inc., Lake Placid, NY).
Two-hybrid Screen and AnalysisThe full-length cDNA of Mena(S) was subcloned into yeast expression vector pGBKT7. The pGBKT7-Mena(S) plasmid was transformed into yeast strain Y190, and then two-hybrid screening was carried out according to the manufacturer's protocol using a GAL4 DNA activation domain fusion library in pGAD10 (MATCHMAKER human leukocyte cDNA library; Clontech). Yeast colonies that grew in medium lacking histidine were picked up, and their -galactosidase activity was assayed on filters. The isolated clones were sequenced. One of the clones encoded AblBP4 (GenBankTM accession number AF001628
[GenBank]
), which corresponds to human Abi-1. We refer to AblBP4 as Abi-1 in this paper.
To examine protein-protein interactions, the cDNA fragment encoding the full-length Mena or one of its deletion mutants was inserted into pGBKT7. The cDNA fragment encoding the full-length Abi-1 or one of its deletion mutants was inserted into pGAD424. All constructs with deletions were generated by PCR and verified by DNA sequencing. Yeast strain Y190 was transformed with bait and prey vectors. Filter and quantitative assays for -galactosidase activity were performed according to the manufacturer's protocols.
Mammalian Expression PlasmidsThe full-length cDNAs for Mena and Mena(S) were subcloned into mammalian expression plasmid pcDNA3. The pcDNA3-Mena and pcDNA3-Mena(S) plasmids were used to express Mena and Mena(S) in 293T cells.
Deletion mutants of Abi-1 were produced by PCR and verified by DNA sequencing. To generate the Abi-1W426K mutant and mutants in which the tyrosine residues of Mena were replaced with phenylalanine ones, oligonucleotide-directed mutagenesis was carried out with a QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Mutations were verified by DNA sequencing.
Mammalian expression plasmids pEBG (a gift from Bruce Mayer) and pFLAG-CMV-2 (Sigma) were used to express proteins fused with an N-terminal GST and FLAG tag, respectively. Expression plasmids pcDNA3 c-Abl (wild type and K290M) and pcDNA3 HA c-Cbl wild type (10) were gifts from David Baltimore and Wallace Langdon, respectively.
Cells293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. Baf3 and LyD9 cells (a gift from Ronald Palacios) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics plus 0.2% conditioned medium from a mouse interleukin-3-overexpressing myeloma cell line (a gift from Hajime Karasuyama) (27).
Abl+/+ and Abl/mouse embryo primary fibroblasts were prepared from Abl+/+ and Abl/ mice, respectively. The two types of mice were obtained by crossing Abl+/ heterozygous mice, which possess a null mutation in the c-abl locus (28) (a gift from Richard Mulligan and Steve Goff). The primary fibroblasts were prepared from embryonic day 16 embryos and immortalized according to the 3T3 method (29). These fibroblasts were used for Western blotting and immunofluorescence analysis. The fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics.
In Vitro Binding AssayFor the expression of fusion proteins, 293T cells plated on 35-mm dishes were transfected with 1 µg of each expression plasmid using LipofectAMINE PLUS reagent (Invitrogen). At 24 h after transfection, the cells were lysed in 300 µl of lysis buffer (25 mM HEPES-KOH (pH 7.2), 150 mM KCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.5 µg/ml leupeptin, 2 µM pepstatin A, 1% Trasylol (aprotinin solution; Bayer), and 1% Triton X-100). The lysate was centrifuged in a microcentrifuge for 10 min at 15,000 rpm to remove insoluble materials. The supernatant was incubated with glutathione-Sepharose 4B (Amersham Biosciences) for 1.5 h with gentle rotation at 4 °C and then washed three times with the lysis buffer. The materials pulled down with the beads were dissociated from the beads by boiling in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. The samples were resolved by SDS-PAGE and immunostained with appropriate antibodies using ECL (Amersham Biosciences).
Phosphorylation Analysis293T cells were transfected with 1 µg of each expression plasmid and then lysed in the lysis buffer containing phosphatase inhibitors (1 mM sodium orthovanadate and 10 mM sodium fluoride). The total amounts of plasmids were balanced with pcDNA3 or pFLAG-CMV-2 empty vector. For detection of the phosphorylation of GST-Mena, the supernatant (50 µl) was incubated with glutathione-Sepharose 4B and then incubated at 4 °C for 1.5 h with gentle rotation. The beads were washed with the lysis buffer containing phosphatase inhibitors, and the materials pulled down with the beads were analyzed by Western blotting with anti-phosphotyrosine antibody. For detection of the phosphorylation of HA-c-Cbl, anti-HA antibody was added to the supernatant (50 µl), followed by incubation for 3 h at 4 °C. Protein G beads were then added to the mixture, followed by incubation at 4 °C for 1.5 h with gentle rotation. The beads were washed and then analyzed as described above.
Immunofluorescence MicroscopyImmunofluorescence microscopy was performed as described previously (30). Briefly, cells plated on fibronectin-coated coverslips were fixed with 4% paraformaldehyde, followed by sequential incubation with primary antibodies and fluorescein isothiocyanate-conjugated or Texas Red-conjugated secondary antibody. Confocal microscopy was performed with an Olympus Fluoview 300 laser-scanning microscope.
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RESULTS |
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To determine whether or not mena(S) mRNA is expressed, we performed PCR analysis using cDNAs from mouse 17-day embryos, mouse brains, and mouse spleens as templates. To distinguish the PCR products for mena and mena(S), primers were designed to encompass the region corresponding to aa 300333 of Mena. The PCR products for mena (438 bp) and mena(S) (336 bp) were observed in all cDNAs examined. The expression of mena(S) was predominant in mouse spleen (Fig. 1B).
To examine the expression of Mena and Mena(S) at the protein level, we generated a rabbit polyclonal antibody against bacterially expressed Mena. Lysates of rat brains, two kinds of mouse B lymphoid cell lines (Baf3 and LyD9), and 293T cells were analyzed by Western blotting with the anti-Mena antibody. In addition, lysates of 293T cells transfected with an expression plasmid encoding Mena or Mena(S) were also examined (Fig. 1C). In accordance with previous results (20), two protein bands corresponding to 140 and 88 kDa were detected for rat brain lysates. Given its expression only in brain, the former species most likely corresponds to a brain-specific form of Mena, Mena(+). On the other hand, the latter is probably Mena, because it was expressed in all types of cells examined and exhibited the same mobility on gels as exogenously expressed Mena in 293T cells. (Note that the 88-kDa band for the lysates of 293T cells transfected with the Mena expression plasmid was more conspicuous than that for the lysates of nontransfected cells.) In addition to the 88-kDa protein, an 80-kDa protein was detected in Baf3 and LyD9 lysates. Since the 80-kDa protein exhibited the same mobility on gels as exogenously expressed Mena(S), the 80-kDa protein is most likely Mena(S). These results suggest that Mena(S) is expressed in B-lymphoid cell lines, which is consistent with the fact that the mena(S) mRNA is predominantly expressed in spleen.
Identification of Novel Mena-binding Proteins Using a Yeast Two-hybrid SystemTo identify molecules that participate in the c-Abl/Mena pathway in mammalian cells, we searched for Mena-binding proteins using a yeast two-hybrid system. We performed screening with Mena(S) as the bait. We expected that screening with Mena(S) would allow us to obtain new binding proteins, because Mena(S) lacks aa 300333 of Mena, which contains the profilin-binding motif. We used a human leukocyte cDNA library because Mena(S) appeared to be expressed in lymphoid cell lines (Fig. 1, A and C). In total, 1.6 x 107 transformants were screened, and 43 positive clones were obtained. The results of sequence analysis of the positive clones are summarized in Table I. Thirty-six positive clones turned out to be zyxin, which has already been reported to bind to Mena (24). Four clones encoded a novel putative small G protein-interacting protein. Characterization of this protein will be described elsewhere. One clone encoded Abi-1, and two clones encoded its variant, E3B1. Abi-1 was originally identified as a c-Abl-binding protein (14). These results raised the possibility that Abi-1 interacts with Mena.
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The EVH1 Domain of Mena Is Involved in the Interaction with Abi-1We characterized the interaction between Mena and Abi-1 using the yeast two-hybrid system. First, we tested whether or not Mena (a nondeletion form) as well as Mena(S) binds to Abi-1. As shown in the upper panel of Fig. 2, both Mena and Mena(S) bound to Abi-1.
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To determine the binding region for Abi-1 on Mena, we constructed deletion mutants of Mena. We divided Mena into four regions, the EVH1 domain (aa 1111), Arg/Leu/Glu-rich region (aa 112238), proline-rich region (aa 239361), and EVH2 domain (aa 362541). The interactions of Abi-1 with the Mena fragments were investigated. As shown in Fig. 2 (middle panel), only the EVH1 domain bound to Abi-1. A mutant lacking the EVH1 domain did not bind to Abi-1.
Next, we defined the region of Abi-1 that is involved in the binding to Mena. Abi-1 contains an SH3 domain and a polyproline structure near the C terminus. We constructed C-terminal deletion mutants of Abi-1. Abi-1 (aa 1391) and (aa 1363) were SH3 deletion mutants. Abi-1 (aa 1331) was a mutant lacking both the SH3 domain and the polyproline structure. Abi-1336361 was a mutant lacking the polyproline structure. The interactions of these mutants with Mena were examined. As shown in the lower panel of Fig. 2, the two SH3 domain deletion mutants, Abi-1 (aa 1391) and (aa 1363), still interacted with Mena. On the other hand, Abi-1 (aa 1331) and Abi-1
336361 did not interact with Mena. We confirmed these results by quantitative
-galactosidase assaying with o-nitrophenyl
-D-galactopyranoside as an indicator. The wild-type Abi-1 and SH3 deletion mutants exhibited similar levels of interaction with Mena (data not shown). These results suggested that the polyproline structure, but not the SH3 domain, of Abi-1 is critical for the interaction with Mena.
Binding of Mena to Abi-1 in Mammalian CellsThe interaction between Mena and Abi-1 was confirmed using a mammalian cell system. GST or GST-Abi-1 was coexpressed with FLAG-tagged Mena in 293T cells. GST or GST-Abi-1 was pulled-down with glutathione beads, and then the coprecipitated FLAG-tagged Mena was detected. As shown in Fig. 3A, FLAG-tagged Mena was coprecipitated with GST-Abi-1 but not with GST. Next, GST-Mena and FLAG-tagged Abi-1 were coexpressed in 293T cells, and lysates were subjected to coprecipitation analysis. As shown in Fig. 3B, FLAG-tagged Abi-1 was coprecipitated with GST-Mena. These results showed that exogenously expressed Mena and Abi-1 interact with each other in 293T cells.
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To determine whether GST-Abi-1 interacts with endogenous proteins, GST-Abi-1 alone was expressed in 293T cells, and then coprecipitation of endogenous proteins with GST-Abi-1 was examined. As shown in Fig. 3C, endogenous Mena and c-Abl were coprecipitated with GST-Abi-1. However, c-Cbl, which is known to be a substrate of c-Abl (10), was not coprecipitated. Another nonreceptor tyrosine kinase, c-Src, was not coprecipitated with GST-Abi-1, either. Overall, these results suggest a specific interaction between Mena and Abi-1 in mammalian cells.
Abi-1 Promotes c-Abl-mediated Phosphorylation of Mena Ena is reported to be a substrate of Drosophila Abl (19). The finding that Mena interacts directly with Abi-1 prompted us to examine whether or not Abi-1 affects the phosphorylation of Mena by c-Abl (Fig. 4A). 293T cells were cotransfected with expression plasmids for c-Abl, FLAG-tagged Abi-1, and GST-Mena. GST-Mena was pulled down from the cell lysates, and its phosphorylation state was investigated by Western blotting with anti-phosphotyrosine antibody. Phosphorylation of GST-Mena was barely detectable when GST-Mena was coexpressed with c-Abl alone (Fig. 4A, lane 3). A dramatic increase in the phosphorylation of GST-Mena was observed when GST-Mena was coexpressed with FLAG-tagged Abi-1 in addition to c-Abl (Fig. 4A, lane 4). A kinase-deficient mutant of c-Abl, K290M, did not phosphorylate GST-Mena even in the presence of FLAG-tagged Abi-1 (Fig. 4A, lane 5), indicating that the kinase activity of c-Abl is required for the phosphorylation of GST-Mena. Reprobing of the filter used for the detection of the phosphorylation state of GST-Mena with an anti-GST antibody showed that comparable levels of GST-Mena existed in all samples (Fig. 4A, bottom panel). These results demonstrate that FLAG-tagged Abi-1 promoted c-Abl-mediated phosphorylation of GST-Mena.
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To assess the specificity of this effect, we used c-Cbl as a substrate for c-Abl (10). Expression plasmids encoding c-Abl, FLAG-tagged Abi-1, and HA-tagged c-Cbl were cotransfected into 293T cells. HA-tagged c-Cbl was immunoprecipitated from the cell lysates with anti-HA antibody, and then its phosphorylation level was examined. The level of phosphorylation of HA-tagged c-Cbl by c-Abl did not significantly change, regardless of the presence or absence of FLAG-tagged Abi-1 (Fig. 4B, lanes 3 and 4). We examined the phosphorylation of p130cas using the same system. Phosphorylation of p130cas by c-Abl was not promoted in the presence of FLAG-tagged Abi-1 (data not shown). These results support the idea that Abi-1 specifically promotes the phosphorylation of Mena by c-Abl kinase.
Effects of Abi-1 Mutants on Phosphorylation of MenaNext, we attempted to determine the Abi-1 domain involved in the promotion of phosphorylation of Mena by c-Abl kinase. Phosphorylation analysis was performed with the deletion mutants of Abi-1 used for the experiments shown in Fig. 2. In addition, we constructed Abi-1W426K, in which a conserved tryptophan residue in the SH3 domain was replaced by a lysine. Each of the expression plasmids encoding Abi-1 mutants was cotransfected with expression plasmids encoding c-Abl and GST-Mena into 293T cells. GST-Mena was precipitated and immunoblotted with the anti-phosphotyrosine antibody (Fig. 5A). The bands on the blot were quantified with NIH Image (Fig. 5B). The level of phosphorylation of GST-Mena in the lysates of cells cotransfected with Abi-1 (aa 1391) (SH3) was lower than that with the full-length Abi-1 (Fig. 5B, lane 2). A similar low phosphorylation level was observed when Abi-1
336361
polyproline and Abi-1W426K mutants were expressed (Fig. 5B, lanes 4 and 5). On the other hand, Abi-1 (aa 1331) (
SH3,
polyproline) did not promote the phosphorylation of GST-Mena by c-Abl (Fig. 5C, lane 6). These results suggested that both the SH3 domain and the polyproline structure of Abi-1 contribute to the promotion of phosphorylation of Mena by c-Abl kinase.
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Tyr-296 of Mena Is the Sole Phosphorylation SiteMena contains six tyrosine residues (aa 16, 38, 70, 87, 296, and 505). To determine which tyrosine residue in Mena is phosphorylated by c-Abl kinase, we replaced each one by phenylalanine. Plasmids encoding c-Abl, FLAG-tagged Abi-1, and GST-Mena mutants were cotransfected into 293T cells. GST-Mena mutants were precipitated, and their phosphorylation levels were measured by Western blotting with anti-phosphotyrosine antibody (Fig. 6A). The wild-type and Y16F, Y38F, Y70F, Y87F, and Y505F mutants were phosphorylated at almost the same level. On the other hand, the Y296F mutant was not phosphorylated, suggesting that Tyr-296 was the site of phosphorylation. The mutant in which five tyrosine residues (Tyr16, Tyr38, Tyr70, Tyr87, and Tyr505) were replaced by phenylalanines was phosphorylated at the same level as the wild-type Mena, whereas the mutant in which all of the tyrosine residues were replaced was not phosphorylated (Fig. 6B). These results indicate that Tyr-296 of Mena is the sole phosphorylation site in Mena for c-Abl kinase. The results of these mutant experiments are summarized in Fig. 6C.
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Abi-1 Is Colocalized with Mena at the Leading EdgeMena is localized to focal adhesions and the leading edge in fibroblasts (20). Although Abi-2 has been reported to be localized to lamellipodia in murine B16F1 melanoma cells (31), its localization has not been precisely compared with that of Mena. We raised a monoclonal antibody against Abi-1, and then the localization of Abi-1 and Mena was compared by double immunostaining. As shown in Fig. 7A, the monoclonal antibody that we raised detected endogenous Abi-1 in lysates of mouse fibroblasts (Abl+/+ and Abl/) and 293T cells as well as ectopically overexpressed FLAG-tagged Abi-1. The two bands observed for the lysates of fibroblasts (Abl+/+) may reflect the phosphorylation of Abi-1. Abi-1 is known to be phosphorylated by Abl kinase (32). These bands were occasionally observed as one fuzzy band (data not shown).
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The distribution of Mena and Abi-1 in mouse embryo fibroblasts (Abl+/+) was analyzed by immunofluorescence microscopy (Fig. 7B, upper panel). Mena is localized at focal contacts and the leading edge, whereas Abi-1 is localized at the leading edge but not focal contacts. A merged image showed the colocalization of the two proteins at the leading edge. Next, we examined whether or not their localization was affected by depletion of c-Abl. We examined the localization of Abi-1 and Mena in c-Abl-deficient fibroblasts. As shown in the lower panel of Fig. 7B, no significant difference was observed in the staining patterns of the two proteins. This indicates that the presence of c-Abl does not affect the localization of either protein. However, the colocalization of Mena and Abi-1 at the leading edge in the fibroblasts is consistent with the present finding that Abi-1 interacts with Mena and may suggest functional collaboration between these molecules for the regulation of the cytoskeleton.
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DISCUSSION |
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The interaction between c-Abl, Abi-1, and Mena would predict the presence of a ternary complex of the three proteins. We sought to detect one using a yeast three-hybrid system. However, no such complex was obtained (data not shown). One plausible explanation for why a stable ternary complex was not observed is that the binding site for c-Abl on Abi-1 is partially occupied by Mena. Our binding studies revealed that the polyproline structure of Abi-1 is critical for the binding to Mena. On the other hand, the SH3 domain of Abi-1 is essential for the binding to c-Abl, and the polyproline structure facilitates the binding (14). Thus, c-Abl may bind to Abi-1 only through the interaction with the SH3 domain when the polyproline structure is occupied by Mena. These interactions may not be enough for the formation of a stable ternary complex but are probably sufficient for the promotion of the phosphorylation of Mena by c-Abl.
It is interesting that the phosphorylation of Mena by c-Abl is promoted to some extent in the presence of Abi-1 mutants lacking either the SH3 domain or the polyproline structure. One explanation for this observation is that Abi-1 may induce conformational changes in c-Abl and/or Mena upon binding to these proteins. The binding of Abi-1 to c-Abl may endow c-Abl with high affinity for the substrate Mena but not other substrates. The binding of Abi-1 to Mena may also affect the conformation of the phosphorylation site of Mena. In this sense, Abi-1 is not just a scaffold for Mena and c-Abl but a regulator that mediates the presentation of Mena to c-Abl by binding to both a substrate and kinase.
It is reasonable to assume that the phosphorylation of Tyr-296 regulates the function of Mena. Given that Tyr-296 is located in the middle of the proline-rich region, the phosphorylation may affect the interaction of Mena with proteins that bind to this region, such as profilin. Phosphorylation of Tyr-296 may also cause a gross structural change in Mena, which affects the functions of domains (EVH1 and EVH2) distant from the phosphorylation site.
The biological significance of Mena(S) is not clear at present. Mena(S) lacks the profilin-binding motif that is located in the proline-rich region of Mena. Mena(S) is expressed in B-lymphocytes predominantly, suggesting that its function may be limited to specific tissues. It has been reported that the proline-rich region of Mena is dispensable for random cell motility but required for intracellular Listeria movement (34). Mena(S) may regulate reorganization of the actin cytoskeleton in certain circumstances. Alternatively, since Mena(S) is probably not able to bind to profilin, it may block Mena/profilin-mediated pathways by competing with the interaction of Mena with Mena-binding proteins. It is noteworthy that the region deficient in Mena(S) is adjacent to the phosphorylation site of Mena. Phosphorylation may have different effects on Mena and Mena(S)
In summary, we have shown that Abi-1 binds to Mena and c-Abl and specifically promotes the phosphorylation of Mena by c-Abl. Our results point to the biochemical significance of the interaction of the three molecules, c-Abl, Abi-1, and Mena, reflecting a signaling pathway possibly downstream of c-Abl kinase.
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FOOTNOTES |
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These authors contributed equally to this work.
¶¶ Present address: Laboratory of Molecular Oncology, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0101, Japan.
¶ To whom all correspondence should be addressed: Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan. Tel.: 81-426-767110; Fax: 81-426-768866; E-mail: tani{at}ls.toyaku.ac.jp.
1 The 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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