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1 Department of Developmental Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan
2 Department of Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan
3 Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima 734-8553, Japan
4 Department of Medicine and Rheumatology, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan
5 Growth Factor Division, National Cancer Center Research Institute, Tokyo, 104-0045, Japan
6 Department of Allergy and Rheumatology, Faculty of Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan
7 Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan
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Abstract |
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exon 2). In comparison to wild-type cells, Cas exon 2/ cells showed reduced motility, which could be due to impaired tyrosine-phosphorylation of FAK and Cas, reduced FAK/Cas/Src/CrkII binding, and also impaired localization of Cas exon 2 to focal adhesions on fibronectin. In addition, to analyze downstream signaling pathways regulated by Cas exon 2, we performed microarray analyses. Interestingly, we found that a deficiency of Cas exon 2 up-regulated expression of CXC Chemokine Receptor-4 and CC Chemokine Receptor-5, which may be regulated by I
B
phosphorylation. These results indicate that the SH3-encoding exon of Cas participates in cell motility, tyrosine-phosphorylation of FAK and Cas, FAK/Cas/Src/CrkII complex formation, recruitment of Cas to focal adhesions and regulation of cell motility-associated gene expression in primary fibroblasts. |
Introduction |
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Physiologically, Cas becomes tyrosine phosphorylated in response to various extracellular stimuli, such as integrin engagement (Nojima et al. 1995; Vuori & Ruoslahti 1995), which recruits Cas from cytoplasm to focal adhesions (Nakamoto et al. 1997). Tyrosine-phosphorylated Cas binds to CrkII, forming a Cas/CrkII complex (Vuori et al. 1996), which subsequently leads to the activation of the Rac–JNK pathway (Dolfi et al. 1998; Kiyokawa et al. 1998a). In addition, over-expression of Cas promotes cell motility, depending on its association with FAK and CrkII (Cary et al. 1998; Klemke et al. 1998).
To clarify biological roles of Cas, we generated Cas-deficient mice (Honda et al. 1998). Cas-deficient embryos died in utero at 12.5 dpc showing marked systemic congestion and growth retardation (Honda et al. 1998). Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscope analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks (Honda et al. 1998). Cas-deficient fibroblasts showed impaired actin stress fiber formation, defects in cell migration, delayed cell spreading and resistance to Src-induced transformation (Honda et al. 1998, 1999). These results demonstrated that Cas is an actin-assembly molecule, which plays an essential role in embryonic development, cytoskeletal organization and Src-induced cellular transformation. Subsequently, to examine the role of each domain of Cas in these processes, we performed a compensation assay by expressing a series of Cas mutants in Cas-deficient fibroblasts (Huang et al. 2002). The results showed that motifs containing YDXP were indispensable for actin cytoskeleton organization and cell migration, suggesting that CrkII-mediated signaling regulates these biological processes (Huang et al. 2002). In contrast, C-terminal SBD was essential for cell migration, Src-induced transformation and membrane localization of Cas, but was dispensable for the organization of actin stress fibers (Huang et al. 2002). Although the above results provided insights in the roles of SD and SBD, the role of the SH3 domain of Cas, which has been shown to associate with various signaling molecules, remains unclear.
To address this issue, we generated mice deficient in Cas exon 2, which produce a truncated Cas protein lacking the SH3 domain. Heterozygous (Cas exon 2+/) mice, which were apparently normal, were intercrossed to produce homozygous (Cas exon 2/) mutants. Cas exon 2/ mice died in utero at 12.5–13.5 dpc and the detailed analysis of the embryonic lethality of the Cas exon 2/ mice is underway and will be published elsewhere. In this report, we established primary fibroblasts from Cas exon 2-deficient embryos and investigated the roles of Cas exon 2 in cellular functions.
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Results |
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/ cells are slower to initiate migration in the wound healing assay
To investigate functional defects caused by Cas exon 2-deficiency, we established primary fibroblasts from Cas exon 2-deficient (Cas exon 2/) embryos. Figure 1A shows the schematic diagram representing Cas exon 2. Cas exon 2 contains the entire SH3 domain and a part of the SD domain containing one YLVP and four YQXP motifs. It encodes 211 amino acids and the predicted molecular weight of Cas exon 2 is about 23 kDa. The expression of Cas exon 2 protein in Cas exon 2/ fibroblasts was detected almost as the expected size by Western blotting using an antibody against Cas, anti-Cas2 (Sakai et al. 1994) (Fig. 1B). Using the fibroblasts, we first performed wound healing cell migration assays. Migratory processes were assessed at 0, 3, 6, 9 and 12 h after wounding (Fig. 2A). Mean percentages of the filled area at each time points are shown in Fig. 2B. Three hours after wounding, Cas exon 2+/+ cells had filled over 30% of the gap and the cells migrating from both ends of the wound had achieved cell–cell contact. In contrast, only 10% of the area was filled by Cas exon 2/ cells. After 6 and 9 h, the migration deficit in Cas exon 2/ cells was less apparent but still present, and at 12 h the gap was almost filled in both types of the cells. This result demonstrated that Cas exon 2/ cells were deficient in ability to migrate, especially in the early phase of the response.
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/ cells show reduced spreading activity on fibronectin (FN)
We next examined the roles of Cas exon 2 in cell attachment, cell adhesion and cell spreading on FN. The morphological changes in Cas exon 2+/+ and Cas exon 2/ cells were observed at 30, 60 and 120 min after plating on FN-coated dishes (Fig. 3A). The mean percentages of flattened cells at each time point are shown in Fig. 3B. The disparity in cell spreading was most apparent 30 min after plating, when more than 70% of the Cas exon 2+/+ cells had already flattened, while only 37% of Cas exon 2/ cells showed a flattened phenotype. The spreading delay in Cas exon 2/ cells continued and was still observed at 120 min. These results demonstrated that Cas exon 2/ cells had a reduced ability to spread on FN.
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Upon FN stimulation, integrin clustering promotes FAK autophosphorylation at Tyr397, which creates a binding site for the SH2 domain of Src (Mitra et al. 2005). FAK/Src binding leads to the conformational activation of Src and results in an activated FAK/Src signaling complex (Schlaepfer et al. 2004), which enhances tyrosine-phosphorylation of Cas (Sakai et al. 1994; Mitra et al. 2005) Tyrosine-phosphorylated Cas binds to CrkII through the SD domain with preference for YDXP motifs (Songyang et al. 1993), which subsequently leads to activate downstream small GTP-binding proteins through C3G (Kiyokawa et al. 1998a,b; Klemke et al. 1998) and plays a key role in cell migration (Fig. 8A).
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/ cells plated on FN were immunoprecipitated with antibodies against either FAK, Src or CrkII and immunoprecipitated proteins were blotted with anti-Cas2. As shown in Fig. 4A, wild-type Cas was associated with FAK, whereas Cas exon 2 could not bind to FAK. By contrast, Fig. 4B shows that Src bound to both Cas and Cas exon 2 at similar levels. We then analyzed possible alteration in CrkII binding to Cas in Cas exon 2/ cells. As shown in Fig. 4C, in Cas exon 2+/+ cells, stable complex formation of Cas and CrkII was detected, whereas in Cas exon 2/ cells, the binding activity of CrkII to Cas exon 2 was significantly reduced. We then analyzed whether the deficiency of Cas exon 2 might affect tyrosine-phosphorylation of Cas. As shown in Fig. 4D, Cas exon 2 was not tyrosine-phosphorylated upon FN stimulation. In addition, we could not detect FAK tyrosine-phosphorylation and FAK/Src binding (Fig. 4E). These results indicated that Cas exon 2-deficiency impaired formation of the FAK/Cas/CrkII complex, tyrosine-phosphorylation of FAK and Cas, and FAK/Src binding upon FN stimulation.
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We compared the subcellular localization of wild-type Cas and Cas exon 2 in primary fibroblasts stimulated by FN. Cas exon 2+/+ and Cas exon 2/ cells grown on FN-coated coverslips were stained with an anti-Cas2. Anti-vinculin (hVIN-1) staining was also performed to identify focal adhesions. As shown in Fig. 5, following FN stimulation, wild-type Cas was recruited to focal adhesions as previously reported (Nakamoto et al. 1997), as demonstrated by the yellow double staining pattern (Fig. 5, left lower panel, indicated by arrows), whereas Cas exon 2 was retained mainly in the cytoplasm and was not concentrated at focal adhesions (Fig. 5, right lower panel). The results indicated that Cas exon 2 is required for the localization of Cas to focal adhesions upon FN stimulation.
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To further characterize the role of Cas exon 2 in intracellular signaling, we performed microarray analyses to investigate alterations in gene expression caused by Cas exon 2-deficiency. RNA samples extracted from Cas exon 2+/+, Cas exon 2+/ and Cas exon 2/ fibroblasts (12.5 dpc, two embryos for each genotype) were subjected to microarray analysis as described in Experimental procedures. Gene expression patterns of Cas exon 2/ fibroblasts were compared with those of Cas exon 2+/+ and Cas exon 2+/ cells. The complete microarray data set is available from the gene expression omnibus (GEO) database (accession no. GSE8357
[NCBI GEO]
). Expressed sequence tags were excluded and genes that showed more than a 3.0-fold change in expression are presented in Table 1. One interesting aspect of the result is that cell migration- and cell adhesion-associated genes, such as chemokine ligands/receptors and thrombospondin, were listed among the genes up-regulated by Cas exon 2-deficiency. We thus confirmed the up-regulation of these genes in Cas exon 2/ fibroblasts by quantitative real-time RT-PCR analysis. The results showed that the expression levels of three genes, CXCR4, CCR5 and thrombospondin 4, were significantly enhanced by Cas exon 2-deficiency. The changes in expression measured by microarray analysis correlated well with data from quantitative real-time RT-PCR analyses (Fig. 6). These results demonstrated that the loss of Cas exon 2 induced expression of CXCR4, CCR5 and thrombospondin 4, genes involved in cell motility in primary fibroblasts.
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B level was augmented in Cas exon 2/ fibroblasts
We then examined the underlying molecular mechanism for the up-regulated expression of CXCR4 and CCR5 in Cas exon 2/ fibroblasts. It is already demonstrated that the extracellular signal-activated transcription factor nuclear factor-B (NF-B) regulates the expression of some chemokine ligands/receptors, including CXCR4 (Helbig et al. 2003; Kukreja et al. 2005) and CCR5 (Kim et al. 2006). Activation of NF-B requires phosphorylation of IB. Thus, we compared phospho-IB levels between Cas exon 2+/+ and Cas exon 2/ fibroblasts. As shown in Fig. 7, the phosphorylation level of IB was significantly augmented in Cas exon 2/ cells as compared to Cas exon 2+/+ cells. These results indicated that that the NF-B signaling pathway was activated by Cas exon 2 deficiency, which would play a role in up-regulated expression of CXCR4 and CCR5 in Cas exon 2/ fibroblasts.
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Discussion |
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exon 2).
As expected from a previous study (Polte & Hanks 1995), we demonstrated that Cas exon 2 lost its ability to bind to FAK but retained the ability to bind to Src, irrespective of FN stimulation (Fig. 4A and B and data not shown). In addition, we found that upon FN stimulation, the binding activity of Cas exon 2 to CrkII was significantly reduced (Fig. 4C). This result seems curious since the YDXP motifs in the SD, that are the preferred binding site to the CrkII SH2 domain when phosphorylated (Songyang et al. 1993), are all conserved in Cas exon 2. To investigate the underlying mechanism, we analyzed tyrosine-phosphorylation of Cas between two types of cells. Upon FN stimulation, Cas was apparently tyrosine-phosphorylated in Cas exon 2+/+ cells as previously reported (Nojima et al. 1995), whereas we could not detect tyrosine-phosphorylation of Cas exon 2 in Cas exon 2/ cells (Fig. 4D). In addition, we examined tyrosine-phosphorylation of FAK, which is the primary event following integrin stimulation. Surprisingly, FAK was not tyrosine-phosphorylated in Cas exon 2/ cells (Fig. 4E). Furthermore, FAK/Src binding was not detected in Cas exon 2/ cells (Fig. 4E), probably owing to impaired tyrosine-phosphorylation of FAK. These results indicate that Cas exon 2 is essential for FAK auto-phosphorylation upon FN. This idea is in line with a previous study, in which Cas lacking SH3 failed to bind to CrkII, which subsequently abolished FAK/Cas/CrkII complex formation as well as FAK auto-phosphorylation by FN (Iwahara et al. 2004). The underlying mechanism for impaired tyrosine-phosphorylation of FAK in Cas exon 2/ cells remains unclear. One possibility is that the constitutive FAK/Cas binding might be essential for conformational change of FAK tyrosine-phosphorylation. Alternatively, the FAK/Cas complex formation might be required for FAK to keep the tyrosine-phosphorylated state. A previous study showed that CrkII knockdown reduces integrin-stimulated FAK Tyr397 autophosphorylation (Iwahara et al. 2004). Therefore, the Cas/CrkII complex may also affect tyrosine-phosphorylation of FAK as an upstream regulator in reverse (Iwahara et al. 2004).
We also compared the intracellular localization of Cas and Cas exon 2 upon FN stimulation. In contrast to Cas to be localized to focal adhesions as previously reported (Nakamoto et al. 1997), no clear localization of Cas exon 2 at focal adhesions was found (Fig. 5). Impaired recruitment of Cas to focal adhesions following FN stimulation was reported in Src-deficient cells (Nakamoto et al. 1997), and Src can be regarded as a recruiting molecule of Cas to focal adhesions (Kaplan et al. 1995; Honda et al. 1999). Although Cas exon 2 and wild-type Cas have comparable binding abilities to bind Src (Fig. 4B), Cas exon 2 was not found in focal adhesions (Fig. 5), indicating that Cas exon 2 plays an essential role in the localization of Cas to focal adhesions upon FN stimulation. This idea is supported by our previous finding that Cas lacking SH3 failed to localize at focal adhesions on FN stimulation when expressed in COS-7 cells (Nakamoto et al. 1997). While the mechanism is not clear, one possibility is that when Cas exon 2 is recruited to focal adhesions by Src, FAK is not tyrosine-phosphorylated and cannot bind to Cas exon 2 and Src, which in turn would allow release of Cas exon 2 from focal adhesions. Another possibility is that the impaired FAK/Src complex leads to reduced activation of Src, which could not recruit Cas exon 2 to focal adhesions. It is possible that impaired tyrosine-phosphorylation of FAK and Cas leads to incomplete formation of FAK/Cas/Src/CrkII complex and impaired localization of Cas to focal adhesions, which resulted in delayed cell migration (Fig. 2) and spreading on FN (Fig. 3). A previous study using the Cas SD mutants and examining their ability to heal the wound revealed that the effective wound healing was achieved by Cas variants containing at least four of the YDXP/YAVP motifs, the major phosphorylation sites of Cas SD (Shin et al. 2004). Since YDXP/YAVP motifs, which serve main binding sites to CrkII, are all conserved in Cas exon 2/ cells (see Fig. 1A), it would be unlikely that the reduced motility is due to the lack of YLVP/YQXP motifs existing in exon 2. In addition, we found that the defects observed in Cas exon 2/ cells were less apparent than those in Cas–/– cells (Honda et al. 1999). The reason might be that since Cas exon 2/ cells retain the YDXP/YAVP motifs and the SBD as compared to Cas–/– cells, these domains would partly participate in downstream signaling. In fact, a slight amount of CrkII could bind to Cas upon FN stimulation (Fig. 4C).
To identify downstream molecules regulated by Cas exon 2, we investigated the expression profile of Cas exon 2/ fibroblasts using microarray methods (Table 1). Interestingly, we found that cell migration- and cell adhesion-associated genes, such as chemokine ligands/receptors and thrombospondin, were up-regulated by Cas exon 2 deficiency. We previously compared the expression profile of Cas–/– fibroblasts with that in Cas-re-expressing fibroblasts using the same methods (Nakamoto et al. 2002), but could not detect changes in expression of chemokine ligands/receptors and thrombospondins. Thus, the expression changes in these genes (chemokine ligands/receptors and thrombospondins) may be specifically regulated by Cas exon 2-mediated signals.
We also demonstrated that the phospho-IB level was augmented in Cas exon 2/ cells, indicating that the NF-B signaling pathway was activated by Cas exon 2 deficiency (Fig. 7). Based on this result, it is conceivable that up-regulated expression of CXCR4 and CCR5 in Cas exon 2/ fibroblasts is, at least in part, dependent on IB phosphorylation. The mechanism underlying the activation of NF-B signaling and up-regulated expression of CXCR4 and CCR5 is not clear. One possibility is that since the 5'-promoter region of FAK contains NF-B binding sites, the NF-B transcription factor might play a role in regulating FAK transcription (Golubovskaya et al. 2004). It would also be possible that NF-B is activated to compensate for the impaired tyrosine-phosphorylation of FAK and FAK/Cas binding in Cas exon 2/ cells.
In summary, we demonstrated that Cas exon 2 plays an essential role in cell migration, cell spreading on FN, tyrosine-phosphorylation of FAK and Cas, FAK/Cas/Src/CrkII complex formation and recruitment of Cas to focal adhesions in primary fibroblasts. In addition, we showed that Cas exon 2-deficiency significantly up-regulated expression of CXCR4 and CCR5, molecules implicated in cell motility (Fig. 8). Our findings define the biological roles of Cas exon 2 and provide novel insights into Cas SH3 function in intracellular signaling.
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Experimental procedures |
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A polyclonal antibody against Cas, anti-Cas2, was generated as previously described (Sakai et al. 1994). Antibodies against FAK, Src and CrkII were from Santa Cruz Biotechnology, Santa Cruz, CA, anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology, Lake Placid, NY and hVIN-1 was from Sigma, St. Louis, MO. Anti-IB and anti-phospho-IB were from Cell Signaling Technology, Danvers, MA. Anti-Fluorescein-labeled and Texas red-labeled secondary antibodies were from Invitrogen, Carlsbad, CA.
Cultivation of primary fibroblasts
Cas exon 2+/ mice were intercrossed and embryos at 12.5 dpc were collected. Heads and internal organs were used for genotyping and primary embryonic fibroblasts were isolated from the remaining of embryos and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 C with 5% CO2. Experiments were performed between three and five passages.
Immunoblotting and immunoprecipitation
Immunoblotting and immunoprecipitation were performed essentially as previously described (Huang et al. 2002). Proteins were extracted by lysing cells in ice-cold 1% Triton lysis buffer [50 mM Tris–HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, 100 mM NaF, 1 mM Na3VO4]. For Western blotting, samples were separated by SDS-PAGE and probed with indicated antibodies. Positive signals were visualized with an enhanced chemiluminescence system (Amersham, Uppsala, Sweden). For immunoprecipitation, 500 µg protein aliquots were incubated with the indicated antibodies for 2 h at 4 C and subsequently with Protein A-Sepharose (Invitrogen) for 1 h at 4 C. Beads were washed 4 times with 1% Triton lysis buffer and boiled in sample buffer prior to SDS-PAGE analysis.
Cell stimulation with FN
Serum-starved cells were removed from the culture dishes by 0.05% trypsin treatment and were resuspended in DMEM. Culture dishes were coated overnight with 10 µg/mL FN (Chemicon, Temecula, CA) at 4 C. The suspended cells were then plated on FN-coated dishes and incubated at 37 C for various periods of time as described previously (Iwahara et al. 2004).
Wound healing cell migration assay
The wound healing cell migration assay was performed according to a method used previously (Honda et al. 1999). In brief, cells were first grown to confluence in plastic culture dishes, and a wound was made in the cell monolayer using a sterile micropipette tip. Then cells were washed 3 times with PBS and cultured at 37 C in DMEM containing 10% FBS. Cell movement was assessed 3, 6, 9 and 12 h after wounding. The percentage of reduced distance between the nuclei of cells at each time period relative to the distance between two rims in the cleared field at the beginning was taken as the index.
Cell spreading assay
The cell spreading assay was performed as previously described (Honda et al. 1999). In brief, serum-starved cells were removed from the culture dishes by exposure to 0.05% trypsin–EDTA, and 2 x 105 cells in a volume of 1 mL DMEM were added to 35 mm tissue culture dishes coated with 10 µg/mL FN. The dishes were incubated at 37 C for the indicated periods of time. Single cells that were phase-bright with rounded morphology were scored as non-spread, whereas those that possessed a flattened shape and looked phase-dark were scored as spread. The number of spread cells was calculated as percentage of the total cells in eight independent fields.
Immunofluorescence
Immunofluorescence was performed as previously described (Nakamoto et al. 1997). Cells were grown on FN-coated coverslips (Matsunami, Osaka, Japan) for 90 min. They were washed 3 times with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS. The fixed cells were washed twice with PBS and permealized with 0.2% Triton-X in PBS. The cells were rinsed and then blocked in PBS plus 3% bovine serum albumin (Sigma). Primary antibodies were used at the following dilutions for 3 h at room temperature in a humidified chamber: 1 : 200 for anti-Cas2, and 1 : 200 for hVIN-1. The coverslips were washed 3 times with PBS and treated with secondary antibodies at the recommended dilutions. After three washes with PBS, the coverslips were mounted in a 1 : 2 mixture of glycerol and PBS. The cells were examined with a LSM5 PASCAL confocal microscopic system (Carl Zeiss, Germany).
Microarray analysis
Total RNA was extracted from primary fibroblasts using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. Two micrograms of total RNA from each sample were labeled using One-cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA) and hybridized with a GeneChip slide (Mouse Genome 430 2.0 Array, Affymetrix). Hybridization was performed at 45 C for 16 h. After hybridization, slides were washed, dried and scanned using the GeneChip Scanner 3000 (Affymetrix). The array results were analyzed using GeneSpring (Agilent Technologies, Santa Clara, CA).
Quantitative real-time RT-PCR analysis
To confirm the differences in expression levels of the genes identified, we used fluorescent-based quantitative real-time RT-PCR with a TaqMan probe. RT-PCR was performed in 20 µL reaction mixtures containing 4 µL of 5 x LightCycler Taqman Master (Roche), 200 nM each primer and 100 nM Universal ProbeLibrary probe (Roche, Basel, Switzerland). Amplification reaction was carried out in a 384-well reaction plate in a spectrofluorimetric thermal cycler (ABI PRISM 7900 Sequence Detector, Applied Biosystems, Foster City, CA). A threshold cycle (Ct) for each sample was calculated by the point in which the fluorescence exceeded the threshold limit. To normalize the samples for loading total RNA equivalent, the second real-time PCR assay was performed targeting the 18S ribosomal RNA gene.
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Acknowledgements |
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This work was in part supported by Grants-in Aids from the Ministry of Education, Culture, Sports, Science and Technology of Japan, from Tsuchiya Foundation, from the Astellas Foundation for Research on Metabolic Disorders, from the Ichiro Kanehara Foundation, and from Hiroshima University 21st Century Center of Excellence Program for Radiation Casualty Medical Research.
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Footnotes |
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* Correspondence: E-mail: hhonda{at}hiroshima-u.ac.jp
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References |
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Accepted: 6 November 2007
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