J Biol Chem, Vol. 275, Issue 9, 6404-6410, March 3, 2000

Chat, a Cas/HEF1-associated Adaptor Protein That Integrates Multiple Signaling Pathways^*

Akira Sakakibara and Seisuke Hattori

From the Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cas (Crk-associated substrate) and HEF1 (human enhancer of filamentation) are related adaptor proteins that function in integrin-mediated cell adhesion and antigen receptor signaling pathways. We report here a molecular cloning of Chat (Cas/HEF1-associated signal transducer) that associates with Cas and HEF1. Chat is a 78-kDa signaling molecule with an N-terminal SH2 domain and is expressed in a wide range of tissues. In hematopoietic cells, a 115-kDa isoform of Chat (Chat-H) was specifically expressed. Chat is associated with Cas in brain, and Chat-H is associated with HEF1 in splenocytes. Deletion analyses revealed that Chat and Cas are associated with each other by their C-terminal domains. Treatment of PC12 cells with epidermal growth factor or nerve growth factor increased the phosphorylation level of Chat. This increase was suppressed by an inhibitor of mitogen-activated protein (MAP) kinase kinase, PD98059, suggesting the phosphorylation of Chat by MAP kinase. In Chat-overexpressed COS7 cells, the activity of c-Jun N-terminal kinase was up-regulated. After the epidermal growth factor stimulation, Chat and Cas were colocalized with actin filaments at ruffling membranes. These findings suggest that Chat transduces signals of tyrosine kinases and MAP kinase to Cas signaling pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell adhesion to extracellular matrix (ECM)¹ concerns various biological events, such as cell growth, survival, differentiation, and migration, which is mediated by integrin receptors in a wide range of cell types (reviewed in Refs. 1 and 2). Integrin family proteins consist of heterodimeric transmembrane proteins and have an ECM-binding extracellular domain and a cytoplasmic domain associating with actin cytoskeleton. At the cytoplasmic surface of integrin-ECM adhesion site, subsets of signaling molecules are recruited and form multi-molecular signaling complex.

Tyrosine kinases play a key role in both the integrin-mediated signaling and the regulation of cell adhesion (reviewed in Refs. 3-5). Several lines of evidence suggest that the stimulation of receptor-type tyrosine kinases and the activation of integrin show a synergistic effect on their downstream events (Refs. 6-8 and reviewed in Ref. 9). Integrin-ECM interaction primarily activates focal adhesion kinase (FAK) (10) and Src (4). This activation in turn triggers tyrosine phosphorylation of several signaling molecules and structural proteins including Cas (11, 12), Shc (13), paxillin (14), cortactin (12), and tensin (15), which further activate downstream signals and promote membrane-cytoskeletal reorganizations.

Protein modules, such as Src homology 2 (SH2) and SH3 domains that respectively bind to tyrosine phosphorylated proteins (16) and proline-rich motifs (17), function in protein-protein interaction in these signaling pathways. Several signaling molecules are constituted of only these modules and their binding motifs, and are termed "adaptor proteins" (reviewed in Refs. 18 and 19).

Cas, originally identified as a major tyrosine phosphorylated protein in v-Crk- or v-Src-transformed fibroblasts, belongs to this adaptor protein family (20). Cas contains, from the N terminus, SH3 domain, substrate domain with multiple SH2 binding sites, and SH2 and SH3 domain-binding motifs for Src family kinases (21). FAK is a binding partner of the SH3 domain (22), and when cells attach to ECM, stimulates Src-mediated tyrosine-phosphorylation of Cas through its interaction with Src (23). Protein-tyrosine phosphatases PTP-1B (24) and PTP-PEST (25) also bind to the same domain and suppress the Cas function through the dephosphorylation of Cas.

Integrin-mediated activation of FAK-Cas signaling pathway elicits the activation of c-Jun N-terminal kinase (JNK), which is necessary for proper cell cycle progression (26). In this pathway, tyrosine-phosphorylated residues in substrate domain of Cas serve as multiple binding sites for another adaptor protein, Crk (27). Crk recruits there its downstream effector, DOCK180 (28, 29), which activates Rac1 and causes subsequent activation of JNK. DOCK180-Rac1 pathway also affects the cytoskeletal reorganization and cell spreading (29). Interestingly, overexpression of Cas promotes the membrane ruffling and cell migration on ECM by a Rac1-dependent manner (30), suggesting a similar signaling cascade. Furthermore, several growth factors stimulate Cas-dependent cell migration (30), which suggests that receptor tyrosine kinases transmit signals to Cas.

HEF1/Cas-L (31, 32) and Efs/Sin (33, 34) are members of the Cas family with common structural features. HEF1 is preferentially expressed in lymphocytes (32) and functions in 1 integrin-mediated signaling and antigen receptor signaling pathways (35, 36). The physiological function of Efs/Sin remains elusive. The most C-terminal regions of these proteins including Cas show significantly high sequence similarity to one another; however, the function of these regions has not been characterized yet.

In this study, we have isolated a gene encoding an SH2 domain-containing adaptor protein, Chat (Cas/HEF1-associated signal transducer), and identified its hematopoietic cell-specific isoform, Chat-H. Chat and Chat-H bound, respectively, to the C-terminal domains of Cas and HEF1. Furthermore, Chat was phosphorylated by mitogen-activated protein (MAP) kinase after growth factor stimulation, and overexpression of Chat caused JNK activation. We also demonstrated that Chat and Cas were colocalized at ruffling membranes. These results suggest that the Chat-Cas complex regulates the JNK signaling pathway and membrane ruffling at the downstream regions of tyrosine kinases and MAP kinase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Cells-- In the process of an immunoscreening of a mouse lung cDNA library constructed in gt11 using a monoclonal antibody B45/18, the initial clone was isolated by a cross-reaction of the antibody.² Using this clone as a probe, full-length mouse cDNA encoding Chat was obtained. After subcloning the insert into pBluescript SK() vector (Stratagene), nucleotide sequencing was performed.

Rabbit anti-Chat polyclonal antibodies (anti-Chat SH, amino acid residues 57-167; anti-Chat CT, residues 308-702; anti-Chat CP, residues 687-702) were raised against the SH2 domain and the C-terminal half-region of Chat produced in Escherichia coli, and a hemocyanin-conjugated synthetic oligopeptide, respectively. Anti-p130^Cas (anti-Cas/HEF1), recognizing both Cas and HEF1, was purchased from Transduction Labolatories. Anti-phosphotyrosine 4G10 was obtained from Upstate Biotechnology. Anti-FLAG M2 (Sigma) and Anti-HA 3F10 (Roche Molecular Biochemicals) were used for epitope-tagged proteins.

PC12 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5% horse serum. COS7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

For growth factor stimulation, PC12 cells were incubated with 50 ng/ml epidermal growth factor (EGF; Takara) or 50 ng/ml nerve growth factor (NGF; Upstate Biotechnology) for 3 min. For PD98059 (Research Biochemicals International) treatment, cells were preincubated with 50 µM PD98059 for 30 min before growth factor stimulation. COS7 cells were serum-starved for 16 h in Dulbecco's modified Eagle's medium supplemented with 0.5% bovine serum albumin before EGF stimulation.

Isolation of Hematopoietic Cells-- Mouse lymphoid tissues dissected in RPMI 1640 medium supplemented with 5% fetal bovine serum were passed through a mesh. The suspended cells were collected by centrifugation at 300 × g for 10 min. For splenocyte isolation, cells were then resuspended in Tris-NH₄Cl solution (0.15 M NH₄Cl, 0.017 M Tris-HCl, pH 7.2) to burst red blood cells. After 2 min at room temperature, the suspension was diluted 10-fold with RPMI 1640 medium and collected by centrifugation at 300 × g for 10 min.

Immunoprecipitation and Immunoblotting-- Mouse tissues were homogenized using a Dounce homogenizer in approximately 20 volumes of ice-cold IP buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 1% Triton X-100, 1 mM Na₃VO₄, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A). After centrifugation at 100,000 × g for 30 min, the supernatants were collected as the tissue lysates. To prepare the lysates from isolated lymphoid cells or cultured cells, cells were lysed with IP buffer and gently rotated for 30 min at 4 °C. The samples were centrifuged at 15,000 × g for 20 min, and supernatants were saved. Appropriate antibodies and protein A-Sepharose (Amersham Pharmacia Biotech) were added to the lysates, and the mixtures were rotated for 3 h at 4 °C. Immunoprecipitates were washed five times with IP buffer and then eluted by boiling in the sample buffer for SDS-polyacrylamide gel electrophoresis for 10 min. Alkaline phosphatase treatment of immunoprecipitates was performed as described (37).

For immunoblotting, proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto a nitrocellulose membrane (Protran, 0.22-µm pore size; Schleicher & Schuell). The membrane was incubated with first antibodies, followed by incubation with horseradish peroxydase-conjugated appropriate second antibodies (Amersham Pharmacia Biotech). Immunoreactive signals were detected with Western blot chemiluminescence reagent (NEN Life Science Products).

Construction of Expression Plasmids and Transfection-- To determine the binding sites for Chat-Cas interaction, expression plasmids for Chat, Cas, and their deletion mutants were constructed. N-terminally FLAG-tagged full-length Chat and a deletion series of Chat (Chat_212-702, Chat_345-702, Chat_346-492, and Chat_1-619) were constructed in pCI-neo vector (Promega), after ligation with an oligonucleotide containing the FLAG sequence. Similaly, full-length Cas and its deleted forms (Cas_425-874, Cas_1-781, and Cas_671-874) were constructed in N-terminal HA-tagging vector pCMV-HA. His-tagged full-length Chat (His-Chat) was constructed by adding six histidine codons upstream the initiation codon of Chat and expressed by using pCAX, a derivative of pCAGGS (38). In JNK assays and immunofluorescence analyses, pCAX was used for the expression of N-terminally FLAG-tagged full-length Chat (pCAX-Chat), and pSSRCas (21) was used for the full-length Cas. For transient expression, transfections were performed using LipofectAMINE plus reagent (Life Tech.) according to the manufacturer's instructions.

In Vitro Binding Assay-- His-tagged Chat was expressed in COS7 cells by transient transfection with pCAX-His-Chat. After 24 h, cells were homogenized in His-binding buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol), and clarified by centrifugation at 15,000 × g for 20 min. His-Bind Resin (Novagen) was mixed with the lysate for 1 h at 4 °C, followed by packaging into a column. The column was washed with His-wash buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10% glycerol) and eluted with His-elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 mM imidazole, 10% glycerol).

To express glutathione S-transferase (GST) fusion protein of Chat association domain of Cas (GST-Cas_671-874) in E. coli, pGEX-4T-1 vector (Amersham Pharmacia Biotech) was used. GST or GST-Cas_671-874 produced in E. coli was bound to glutathione-Sepharose (Amersham Pharmacia Biotech) in Binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol). His-Chat produced in COS7 cells was diluted ten-fold with Binding buffer and incubated with GST-bound or GST-Cas_671-874-bound glutathione-Sepharose for 3 h at 4 °C. After washing five times, the adsorbed materials were subjected to immunoblot with anti-Chat CP.

JNK Assay-- COS7 cells grown on 60-mm dishes were transfected with 1.25 µg each of indicated plasmids as described above. Total amount of transfected DNA was adjusted to 2.5 µg/60-mm dish with control vector pCAX. After 24 h, the cells were serum-starved for 24 h at 37 °C. Preparation of cell lysates and solid phase JNK assay were carried out according to Hibi et al. (39). Briefly, endogenous JNK was pulled down by GST-c-Jun_1-79-coupled glutathione-Sepharose and was subjected to kinase reaction in the presence of [-³²P]ATP. Incorporated radioactivity was quantitated by measuring the signal intensity of the autoradiogram of the samples on SDS-polyacrylamide gel electrophoresis.

Immunofluorescence Microscopy-- For indirect immunofluorescence microscopy, cells were cultured on cover glasses and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. After permeabilization with 0.2% Triton X-100 in PBS for 15 min, cells were soaked in a blocking solution (PBS containing 1% bovine serum albumin) for 30 min. The specimens were incubated with first antibodies for 60 min in a moist chamber, washed three times with the blocking solution, and then incubated for 60 min with the second antibody, Cy2-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-mouse IgG (Amersham Pharmacia Biotech). To visualize actin filaments, Texas Red-conjugated phalloidin (Molecular Probes) was used. Samples were then washed with PBS for three times, mounted in Perma Fluor (Immunon), and examined using a fluorescence microscope, Zeiss Axiophot II photomicroscope (Carl Zeiss).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Cloning and Expression Profile of Chat-- In the process to identify an antigen that localizes at cell-cell adherence junctions by immunoscreening of an expression library, we isolated a cDNA clone encoding a novel adaptor protein by a cross-reaction of the antibody. This protein had an N-terminal SH2 domain, and its calculated molecular mass was 78 kDa (Fig. 1, A and B). The central portion of this protein was rich in serine, threonine, and proline residues, and this portion contained four consensus sites for MAP kinase phosphorylation (Pro-X-Ser/Thr-Pro) shown by double underlines in Fig. 1A. As described below, this protein was associated with Cas family adaptor proteins, Cas and HEF1, through its C-terminal domain (Fig. 1B). Considering these properties, we named this protein Chat.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Primary structure and expression profile of Chat. A, the amino acid sequence of mouse Chat deduced from the nucleotide sequence of Chat cDNA (GenBankTM accession number AB030442). The SH2 domain is underlined. MAP kinase phosphorylation consensus sites are shown by double underlines. The Cas association domain that is revealed by this study is indicated in the left side of the sequence. B, schematic structure of Chat. The SH2 domain and Cas association domain are represented. C, immunoblot analysis using anti-Chat CP antibody. Lysates of various mouse tissues and hematopoietic cells prepared from lymphoid tissues (30 µg each) were loaded.

As the first step, we examined the expression of Chat in various tissues of mouse. Immunoblot analysis using anti-Chat CP polyclonal antibody showed a relatively ubiquitous expression pattern of Chat at the molecular mass of 78 kDa (Fig. 1C, arrow). A 115-kDa isoform of Chat was specifically expressed in hematopoietic cells from spleen, lymph node, and thymus (Fig. 1C, arrowhead). Although the detailed molecular nature of this 115-kDa protein remained to be clarified, we tentatively named the isoform Chat-H, meaning the hematopoietic cell-specific Chat. Same results were obtained with other anti-Chat polyclonal antibodies that recognized distinct epitopes (anti-Chat SH and anti-Chat CT; data not shown).

Immunoblot analysis of various established cell lines showed that Chat was expressed in a wide range of cell lines at different levels (data not shown). Only Chat-H species was detected in hematopoietic cell lines (WEHI 231, BAL17, HL-60, Jurkat, etc.; data not shown), which confirmed the result obtained with tissue extracts.

Identification of Cas and HEF1 as Chat-associated Partners-- To seek for a clue to Chat function, we tried to identify a tyrosine phosphorylated protein that formed a complex with Chat, because Chat had one SH2 domain at its N terminus. Chat was immunoprecipitated with anti-Chat-CT from lysates of mouse brain and splenocytes, and immunoprecipitates were subjected to immunoblot analyses (Fig. 2). Anti-Chat-immunoblot confirmed the results in the previous section that brain and lymphoid cells expressed only Chat and Chat-H, respectively (Fig. 2, top panel). Tyrosine-phosphorylated proteins with apparent molecular masses of 130 kDa (in brain) and 115 kDa (in spleen) were detected as major Chat-associated molecules (Fig. 2, middle panel).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Identification of Chat-associated tyrosine-phosphorylated proteins. Whole mouse brain extract or splenocyte cell lysate was subjected to immunoprecipitation (IP) using anti-Chat CT antibody or control rabbit IgG, and then the immunoprecipitates were analyzed by immunoblot with anti-Chat CP (top panel), anti-phosphotyrosine (middle panel), or anti-Cas/HEF1 (bottom panel) antibody.

We then applied antibodies against various known tyrosine-phosphorylated proteins, the molecular mass of which was between 115 and 130 kDa. An anti-Cas antibody that recognized Cas and HEF1 reacted with both Chat-associated proteins (Fig. 2, bottom panel). This result revealed that the 130-kDa protein and the 115-kDa protein were Cas and HEF1, respectively.

Determination of Binding Sites of Chat and Cas for Chat-Cas Interaction-- The essential region of Chat for the association of Cas was determined by co-immunoprecipitation assay using various deletion mutants of Chat and full-length Cas coexpressed in COS7 cells (Fig. 3A). As shown in Fig. 3B, full-length Chat, Chat_212-702 and Chat_345-702 associated with Cas to a similar extent, indicating that the C-terminal half-region of Chat (Chat_345-702) was fully capable of the Chat-Cas interaction. However, the deletion of 146 amino acid residues from the middle (Chat_346-492) or 78 residues from the C terminus (Chat_1-619) completely disrupted the binding activity. The expression level of these proteins and Cas were similar among these samples (Fig. 3B). These results indicate that the C-terminal half-region of Chat but not the SH2 domain is required for the association with Cas, and we named the C-terminal region "Cas association domain" (Fig. 3A).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Chat forms a complex with Cas through its C-terminal domain. FLAG-tagged Chat or its deletion mutants (schematically shown in A) and Cas expression plasmids were co-transfected into COS7 cells. After 24 h, cells were lysed, and FLAG-tagged Chat was immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblot with anti-Cas/HEF1 (B, top panel). The expression levels of Chat and its derivatives (middle panel), and Cas (bottom panel) were analyzed by immunoblots with anti-FLAG and anti-Cas/HEF1 by using total lysates of the same transfectants.

Cas is known to form a complex with diverse signaling molecules through its SH3, substrate, and Src binding domains (20-22, 24, 25). To identify the binding site on Cas for the association with Chat, we then performed co-immunoprecipitation assay using a series of deletion mutants of Cas and full-length Chat (Fig. 4). N-terminally deleted mutants (Cas_425-874 and Cas_671-874) still fully retained the association capacity with Chat (Fig. 4B), demonstrating that the C-terminal 204 residues are sufficient for the association. By the deletion of C-terminal 93 residues, however, Cas completely lost the association activity (Cas_1-781, in Fig. 4B). Therefore, we defined the C-terminal 204 residues of Cas as "Chat association domain" (Fig. 4A).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Cas C-terminal domain binds to Chat. Chat association domain of Cas was determined using various HA-tagged Cas constructs (A) and Chat expression plasmid as in Fig. 3. B, Chat recovered in anti-HA immunoprecipitates (IP) was analyzed by anti-Chat CP immunoblot. C, His-tagged Chat produced in COS7 cells was pulled down with GST-bound or GST-Cas671-874-bound glutathione-Sepharose beads. The trapped Chat was detected with anti-Chat CP antibody.

To examine whether the association of Chat and Cas was direct or not, we carried out in vitro binding experiment. N-terminally histidine-tagged full-length Chat was expressed in COS7 cells and partially purified by a nickel affinity column. The purified protein was mixed with GST or GST-Cas_671-874 that had been immobilized on glutathione-Sepharose beads. As shown in Fig. 4C, only GST-Cas_671-874 pulled down Chat, suggesting that Chat-Cas interaction is direct.

Chat Phosphorylation Induced by EGF or NGF in PC12 Cells-- To clarify the upstream signaling pathway of Chat, we examined whether endogenous Chat in PC12 cells underwent phosphorylation during the treatment of cells with either EGF or NGF (Fig. 5A). Both EGF and NGF caused upward electrophoretic mobility shift of Chat to a similar extent (top panel). However, Chat in these cells was not recognized by an anti-phosphotyrosine antibody, indicating that Chat was not tyrosine phosphorylated under the condition (middle panel). When anti-Chat immunoprecipitates were treated with calf intestine alkaline phosphatase, the mobility of Chat from both control and EGF-treated cells increased to a same level (top panel). This result indicated that Chat was phosphorylated to some extent in the control cells and that the phosphorylation level further increased by EGF treatment. Because Chat before the phosphatase treatment was detected as a significantly broad band, multiple sites might be phosphorylated. Even after the phosphatase treatment, Cas still bound to Chat, which suggested that the Chat-Cas interaction was phosphorylation-independent (bottom panel).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Chat is phosphorylated after EGF or NGF stimulation in PC12 cells. A, PC12 cells were stimulated with 50 ng/ml EGF (+EGF) or 50 ng/ml NGF (+NGF) for 3 min at 37 °C or left untreated (Control). Anti-Chat CT immunoprecipitates were subjected to immunoblot with anti-Chat CP (top panel), anti-phosphotyrosine (middle panel), or anti-Cas/HEF1 (bottom panel). In the right-hand panels, the immunoprecipitates were incubated in the presence (+AP) or absence (AP) of alkaline phosphatase with (+PI) or without phosphatase inhibitors prior to immunoblot. B, PC12 cells were treated with 50 µM PD98059 (+PD98059) or a control solvent (+DMSO) for 30 min at 37 °C, followed by stimulation with 50 ng/ml EGF (+EGF) for 3 min. Anti-Chat CP immunoblot was carried out as in A.

Because Chat had four potential MAP kinase phosphorylation sites (Fig. 1A), we examined the effect of an inhibitor of MAP kinase and/or ERK2 kinase, PD98059, on Chat phosphorylation induced by EGF. PD98059 treatment clearly suppressed the upward shift of Chat (Fig. 5B), suggesting that MAP kinase is a main kinase responsible for the EGF-induced Chat phosphorylation.

Activation of JNK by Chat Overexpression-- Recent studies reported that JNK is up-regulated when a member of Cas signaling pathway is overexpressed (29, 40). To assess the possibility that Chat is also involved in this signaling pathway, we measured JNK activity of COS7 cells overexpressing Chat (Fig. 6). In Chat-overexpressing cells, JNK activity was 1.7-fold higher than in the control cells (Fig. 6B). This increase was similar to that induced by Cas (1.9-fold (Fig. 6B) or the data in Ref. 40) or Crk (40). Coexpression of Chat and Cas activated JNK to a similar level (2.0-fold (Fig. 6B)). The amount of JNK did not vary significantly among the samples (data not shown). These results suggest that Chat is also involved in the Cas-mediated JNK activation pathway.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of Chat and Cas overexpression on JNK activation. COS7 cells were transfected in duplicate with pCAX (Control), pCAX-Chat (Chat), pSSRCas (Cas), or a mixture of pCAX-Chat and pSSRCas (Chat+Cas). 2 days after transfection, endogenous JNK activity was determined according to Hibi et al. (39). A, GST-c-Jun1-79-precipitated JNK activity was visualized by autoradiography of 32P-labeled GST-c-Jun1-79 on SDS-polyacrylamide gel electrophoresis. Duplicate samples (#1 and #2) are shown. B, JNK activity was quantified from digitized image of the autoradiogram by using National Institutes of Health IMAGE analysis program. The values are illustrated relatively taking the value of control cells as 1. Each value (an average of the duplicate samples) represents the mean ± S.E. of three independent experiments. Each transfectants showed statistically significant increase in relative activity over control cells (Chat, p < 0.01; Cas, p < 0.005; and Chat+Cas, p < 0.001) as determined by Student's test.

Colocalization of Chat and Cas in Living Cells-- To further confirm the association of Chat with Cas, we performed immunofluorescence analyses of COS7 cells expressing Chat together with staining of actin cytoskeletal structures (Fig. 7). In Chat-transfected COS7 cells, Chat was predominantly detected throughout the cytoplasm (Fig. 7A). Rapid translocation of Chat to ruffling membranes was observed after stimulation of membrane ruffling by EGF (Fig. 7, C and D) or insulin (data not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Chat is colocalized with the actin filament and Cas at ruffling membranes. COS7 cells were transfected with either FLAG-tagged Chat alone (A-D) or in combination with Cas (E-H). After 24 h, cells were serum-starved for 16 h. Then the cells were fixed after 15 min of treatment with (C, D, G, and H) or without 50 ng/ml EGF (A, B, E, and F). Immunofluorescense analyses using anti-FLAG (A and C), anti-Chat SH (E and G), or anti-Cas (F and H) antibody are shown. For visualization of actin filaments, Texas Red-conjugated phalloidin was used (B and D). Chat was detected throughout the cytoplasm, and concentrated localization was observed at ruffling membranes (arrowheads). Bar, 10 µm.

Chat and Cas were then expressed simultaneously, and their cellular distributions were studied. In unstimulated cells, both Chat (Fig. 7E) and Cas (Fig. 7F) were mainly localized in the cytoplasm. When cells were EGF-stimulated, intense staining of Chat (Fig. 7G) and Cas (Fig. 7H) at ruffling membranes was observed, and their patterns were very similar to each other. Taken together with the results that Chat and Cas formed a complex in cell lysates, the association of Chat and Cas in living cells was strongly suggested.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study demonstrates that Chat, an adaptor protein having an SH2 domain, interacts with the C-terminal domain of Cas family adaptor proteins. Chat was phosphorylated by MAP kinase after the stimulation of tyrosine kinase receptors in PC12 cells. In Chat-overexpressing COS7 cells, the JNK activity was up-regulated. Chat showed colocalization with Cas at ruffling membranes. These results suggest that Chat integrates signals from tyrosine kinases and MAP kinase, controlling cell growth and cytoskeletal reorganization through the Cas signaling pathway.

Very recently, Lu et al. (41) reported a molecular cloning of NSP family proteins, NSP1, NSP2, and NSP3, and it emerged that NSP3 is the human ortholog of mouse Chat (90% identity between NSP3 and Chat amino acid sequences; 35-40% identity between Chat and NSP1 or NSP2). BCAR3 that confers antiestrogen resistance to a mammary cell line (42) is identical to NSP2. Ascidian HrSH2 may also be a member of this family (43). As described here Chat/NSP3 binds to Cas. Lu et al. (41) described the Cas-NSP1 interaction in the same study, although they speculated that the association was mediated by Cas SH3 domain. Furthermore, NSP1 undergoes tyrosine phosphorylation and Chat/NSP3 becomes Ser/Thr phosphorylated upon growth factor stimulation. Therefore, these family proteins may participate in novel signaling pathways to Cas.

Tissue immunoblot analyses showed that Chat was expressed ubiquitously except hematopoietic cells, wherein only a hematopoietic cell-specific isoform, Chat-H, was expressed (Fig. 1C). Chat and Chat-H respectively associates with Cas and HEF1 (Fig. 2). HEF1 is tyrosine phosphorylated when B or T cell antigen receptor is stimulated (32, 35, 36). Chat-H was recovered in anti-phosphotyrosine immunoprecipitates when B cell antigen receptor was cross-linked,² consistent with the complex formation of Chat-H with HEF1.

By using various deletion mutants of Chat and Cas, we demonstrated that C-terminal half of Chat binds to C-terminal 204 amino acids of Cas (Figs. 3 and 4). Although the binding of Chat-H to HEF1 was not fully characterized yet, it was highly likely that complex formation between Chat-H and HEF1 was mediated by their C-terminal domains, because (a) our preliminary characterization of Chat-H revealed that Chat and Chat-H share same C-terminal sequence² and (b) the C-terminal region of human HEF1 shows relatively high sequence similarity to Chat association domain of rat Cas (56% identical, 73% conservative among 204 residues). Whether Chat or Chat-H interacts with the third member of Cas family protein, Efs/Sin, remains to be clarified. Similarity between Efs/Sin and Cas (42% identical, 57% conservative among C-terminal 147 residues) is lower both in length and extent compared with that between Cas and HEF1.

Cas family proteins participate in multiple biological events including integrin-mediated cell-matrix adhesion (11, 44), JNK activation (26, 40), cell migration (30), B and T cell antigen receptor signaling (32, 35, 36), and Src-induced transformation (44). Recently, the molecular basis for these events has been intensely investigated. In integrin-mediated cell adhesion, Cas forms a multivalent scaffold complex at focal adhesion sites. This complex includes at least FAK, Src, and Crk as direct binding partners of SH3, Src binding, and substrate domains of Cas, respectively (20-22). The C-terminal regions of Cas family proteins are highly conserved in their primary structures; however, the function of these regions has not been characterized, except that C-terminal portion of HEF1, when expressed in budding yeast, causes the filamentous cell morphology (31). Our result, that Chat binds to the C-terminal domain of Cas (Fig. 4C) and probably to HEF1, reveals a novel function of these domains.

We showed that the phosphorylation level of Chat was elevated when PC12 cells were stimulated by EGF or NGF and suggested MAP kinase as a major kinase responsible for this increase (Fig. 5). The result coincided with the molecular feature of Chat having four MAP kinase phosphorylation sites (Pro-X-Ser/Thr-Pro) in the central region (Fig. 1A). Human NSP3 has five MAP kinase sites in the corresponding region. Interestingly, NSP1 does not have any MAP kinase site in the molecule. It is intriguing to clarify the physiological function of MAP kinase phosphorylation of Chat. Although endogenous Chat in PC12 cells did not undergo tyrosine phosphorylation by EGF (Fig. 5), when overexpressed in COS7 cells, Chat was tyrosine phosphorylated during the stimulation with the same ligand (data not shown). Lu et al. (41) also reported that NSP1 was tyrosine phosphorylated by EGF and that NSP1 bound to activated EGF receptor in a similar overexpression experiment.

Recently, the physiological significance of FAK-Cas signaling pathway in integrin-mediated JNK activation and cell cycle progression was emphasized (26), and several reports demonstrated that overexpression of Cas, or component of Cas signaling pathway, Crk, DOCK180 or NSP1, activates JNK (29, 40, 41). In Chat overexpressing COS7 cells, JNK activity was up-regulated to a similar extent as in Cas overexpressing cells (Fig. 6), suggesting that Chat is also involved in the Cas-mediated JNK activation pathway.

Cas functions as well in the integrin-mediated cytoskeletal reorganization that accompanies with cell adhesion (45) and cell migration (30). In Chat and Cas expressing COS7 cells, both Chat and Cas were concentrated at ruffling membranes after EGF or insulin stimulation (Fig. 7). Ruffling membranes are characteristic of migrating cells, and both EGF and insulin are known to induce cell migration on ECM (30), suggesting that Chat function in these growth factors induced cell migration.

We report here a unique biological feature of Chat integrating signals from tyrosine kinase and MAP kinase. The next important issues that we should resolve are to identify the binding partner of Chat SH2 domain, to clarify the mechanism of Chat translocation to ruffling membranes, and to reveal the effect of MAP kinase-mediated phosphorylation on Chat function. Studies on these lines will provide us further information on the molecular mechanism of how these integrated signals regulate the function of Cas signaling pathway.

	ACKNOWLEDGEMENTS

Initial phase of this project was carried out in Dr. S. Tsukita's laboratory. We thank S. Tsukita for the permission to use the cDNA clone encoding Chat and T. Nakamoto and H. Hirai for providing the Cas expression construct. We also thank S. Nakamura, T. Katagiri, Y. Ohta, Y. Imai, and M. Matsuda for valuable comments and technical advice.

	FOOTNOTES

^* This work was supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB030442.

To whom correspondence should be addressed: Division of Biochemistry and Cellular Biology, National Inst. of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. Tel.: 81-42-346-1722; Fax: 81-42-346-1752; E-mail: hattori@ncnp.go.jp.

² A. Sakakibara, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; FAK, focal adhesion kinase; SH2 and SH3, Src homology 2 and 3; EGF, epidermal growth factor; NGF, nerve growth factor; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; GST, glutathione S-transferase; HA, hemagglutinin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1.	Hynes, R. O. (1992) Cell 69, 11-25[CrossRef][Medline] [Order article via Infotrieve]
2.	Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve]
3.	Parsons, T. (1996) Curr. Opin. Cell Biol. 8, 146-152[CrossRef][Medline] [Order article via Infotrieve]
4.	Hanks, S. K., and Polte, T. R. (1997) BioEssays 19, 137-145[CrossRef][Medline] [Order article via Infotrieve]
5.	Schoenwaelder, S. M., and Burridge, K. (1999) Curr. Opin. Cell Biol. 11, 274[CrossRef][Medline] [Order article via Infotrieve]
6.	Miyamoto, S., Teramoto, H., Gutkind, J. S., and Yamada, K. M. (1996) J. Cell Biol. 135, 1633-1642[Abstract/Free Full Text]
7.	Renshaw, M. W., Ren, X.-D., and Schwartz, M. A. (1997) EMBO J. 16, 5592-5599[CrossRef][Medline] [Order article via Infotrieve]
8.	Woodard, A. S., García-Cardea, G., Leong, M., Madri, J. A., Sessa, W. C., Languino, L. R. (1998) J. Cell Sci. 111, 469-478[Abstract]
9.	Schwartz, M. A., and Baron, V. (1999) Curr. Opin. Cell Biol. 11, 197-202[CrossRef][Medline] [Order article via Infotrieve]
10.	Guan, J. L., and Shalloway, D. (1992) Nature 358, 690-692[CrossRef][Medline] [Order article via Infotrieve]
11.	Nojima, Y., Morino, N., Miura, T., Hamasaki, K., Furuya, H., Sakai, R., Sato, T., Tachibana, K., Morimoto, C., Yazaki, Y., and Hirai, H. (1995) J. Biol. Chem. 270, 15398-15402[Abstract/Free Full Text]
12.	Vuori, K., and Ruoslahti, E. (1995) J. Biol. Chem. 270, 22259-22262[Abstract/Free Full Text]
13.	Schlaepfer, D. D., Jones, K. C., and Hunter, T. (1998) Mol. Cell. Biol. 18, 2571-2585[Abstract/Free Full Text]
14.	Burridge, K., Turner, C. E., and Romer, L. H. (1992) J. Cell Biol. 119, 893-903[Abstract/Free Full Text]
15.	Bockholt, S. M., and Burridge, K. (1993) J. Biol. Chem. 268, 14565-14567[Abstract/Free Full Text]
16.	Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1992) Cell 72, 767-778
17.	Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993) Science 259, 1157-1161[Abstract/Free Full Text]
18.	Schlessinger, J. (1994) Curr. Opin. Genet. Dev. 4, 25-30[CrossRef][Medline] [Order article via Infotrieve]
19.	Birge, R. B., Knundsen, B. S., Besser, D., and Hanafusa, H. (1996) Genes Cells 1, 595-613[Abstract]
20.	Sakai, R., Iwamatsu, A., Hirano, N., Ogawa, S., Tanaka, T., Mano, H., Yazaki, Y., and Hirai, H. (1994) EMBO J. 13, 3748-3756[Medline] [Order article via Infotrieve]
21.	Nakamoto, T., Sakai, R., Ozawa, K., Yazaki, Y., and Hirai, H. (1996) J. Biol. Chem. 271, 8959-8965[Abstract/Free Full Text]
22.	Polte, T. R., and Hanks, S. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10678-10682[Abstract/Free Full Text]
23.	Schlaepfer, D. D., Broome, M. A., and Hunter, T. (1997) Mol. Cell. Biol. 17, 1702-1713[Abstract]
24.	Liu, F., Sells, M. A., and Chernoff, J. (1998) Curr. Biol. 8, 173-176[CrossRef][Medline] [Order article via Infotrieve]
25.	Garton, A. J., Burnham, M. R., Bouton, A. H., and Tonks, N. K. (1997) Oncogene 15, 877-885[CrossRef][Medline] [Order article via Infotrieve]
26.	Oktay, M., Wary, K. K., Dans, M., Birge, R. B., and Giancotti, F. G. (1999) J. Cell Biol. 145, 1461-1469[Abstract/Free Full Text]
27.	Matsuda, M., and Kurata, T. (1996) Cell Signal. 8, 335-340[CrossRef][Medline] [Order article via Infotrieve]
28.	Hasegawa, H., Kiyokawa, E., Tanaka, S., Nagashima, K., Gotoh, N., Shibuya, M., Kurata, T., and Matsuda, M. (1996) Mol. Cell. Biol. 16, 1770-1776[Abstract]
29.	Kiyokawa, E., Hashimoto, Y., Kobayashi, S., Sugimura, H., Kurata, T., and Matsuda, M. (1998) Genes Dev. 12, 3331-3336[Abstract/Free Full Text]
30.	Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K., and Cheresh, D. A. (1998) J. Cell Biol. 140, 961-972[Abstract/Free Full Text]
31.	Law, S. F., Estojak, J., Wang, B., Mysliwiec, T., Kruh, G., and Golemis, E. A. (1996) Mol. Cell. Biol. 16, 3327-3337[Abstract]
32.	Minegishi, M., Tachibana, K., Sato, T., Iwata, S., Nojima, Y., and Morimoto, C. (1996) J. Exp. Med. 184, 1365-1375[Abstract/Free Full Text]
33.	Ishino, M., Ohba, T., Sasaki, H., and Sasaki, T. (1995) Oncogene 11, 2331-2338[Medline] [Order article via Infotrieve]
34.	Alexandropoulos, K., and Baltimore, D. (1996) Genes Dev. 10, 1341-1355[Abstract/Free Full Text]
35.	Manié, S. N., Beck, A. R., Astier, A., Law, S. F., Canty, T., Hirai, H., Druker, B. J., Avraham, H., Haghayeghi, N., Sattler, M., Salgia, R., Griffin, J. D., Golemis, E. A., and Freedman, A. S. (1997) J. Biol. Chem. 272, 4230-4236[Abstract/Free Full Text]
36.	Ohashi, Y., Tachibana, K., Kamiguchi, K., Fujita, H., and Morimoto, C. (1998) J. Biol. Chem. 273, 6446-6451[Abstract/Free Full Text]
37.	Sakakibara, A., Furuse, M., Saitou, M., Ando-Akatsuka, Y., and Tsukita, S. (1997) J. Cell Biol. 137, 1393-1401[Abstract/Free Full Text]
38.	Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193-200[CrossRef][Medline] [Order article via Infotrieve]
39.	Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes Dev. 7, 2135-2148[Abstract/Free Full Text]
40.	Dolfi, F., Garcia-Guzman, M., Ojaniemi, M., Nakamura, H., Matsuda, M., and Vuori, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15394-15399[Abstract/Free Full Text]
41.	Lu, Y., Brush, J., and Stewart, T. A. (1999) J Biol. Chem. 274, 10047-10052[Abstract/Free Full Text]
42.	van Agthoven, T., van Agthoven, T. L. A., Dekker, A., van der Spek, P. J., Vreede, L., and Dorssers, L. C. J. (1998) EMBO J. 17, 2799-2808[CrossRef][Medline] [Order article via Infotrieve]
43.	Takahashi, H., and Satoh, N. (1998) Dev. Growth Differ. 40, 431-438[CrossRef][Medline] [Order article via Infotrieve]
44.	Honda, H., Oda, H., Nakamoto, T., Honda, Z.-I., Sakai, R., Suzuki, T., Saito, T., Nakamura, K., Nakao, K., Ishikawa, T., Katsuki, M., Yazaki, Y., and Hirai, H. (1998) Nat. Genet. 19, 361-365[CrossRef][Medline] [Order article via Infotrieve]
45.	Nakamoto, T., Sakai, R., Honda, H., Ogawa, S., Ueno, H., Suzuki, T., Aizawa, S.-I., Yazaki, Y., and Hirai, H. (1997) Mol. Cell. Biol. 17, 3884-3897[Abstract]