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doi: 10.1242/10.1242/jcs.00207

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Novel function of Chat in controlling cell adhesion via Cas-Crk-C3G-pathway-mediated Rap1 activation

Akira Sakakibara1,*,§, Yusuke Ohba2, Kazuo Kurokawa2, Michiyuki Matsuda2 and Seisuke Hattori1,{ddagger}

1 Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan
2 Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
* Present address: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA
{ddagger} Present address: Division of Cellular Proteomics, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan



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  		Fig. 1. Rap1 activation induced by overexpression of Chat. (A) A diagram of Chat variants used in this study. We used an N-terminally myristoylated membrane-targeted form of Chat (Myr-Chat) to evaluate the activation status of Chat. C-terminal deletion mutants (Chat-{Delta}CT and Myr-{Delta}CT) lacked the Cas association activity. (B) 293T cells were transiently transfected with the indicated Chat-related expression plasmid together with small GTPase Rap1. After 30 hours, the activation level of Rap1 was compared by a pull-down assay. The amount of pulled-down GTP-bound Rap1 (GTP-Rap1), expression levels of transfected Rap1 (Total Rap1) and Chat-derivatives (Anti-Chat) is shown. Overexpression of Chat increased the GTP-bound Rap1 compared with the control pCAX vector transfectant. Myr-Chat activated Rap1 more effectively than wild-type Chat. Chat-{Delta}CT and Myr-{Delta}CT abolished the Rap1 activation competence.

 


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  		Fig. 2. Effect of Chat overexpression on Ras family small GTPase activities. Activation levels of the small GTPases in Chat-overexpressing 293T cells were examined by a pull down assay as in Fig. 1. (A) Overexpression of Chat or Myr-Chat induced Rap1 activation. The activation level was low compared with a positive control Rap1 GEF, C3G. Neither Chat nor Myr-Chat increased the amount of GTP-bound H-Ras (B), R-Ras (C) and RalA (D) in similar experiments to A. mSos-F, a membrane-targeted form of mSos, C3G or RalGDL was used as a positive control for H-Ras, R-Ras or RalA activation.

 


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  		Fig. 3. Overexpression of the Chat-Cas-Crk pathway member activates Rap1. (A) Overexpression effect of Chat and Cas on Rap1 activation was examined by a pull-down assay of GTP-bound Rap1 as in Fig. 1. Overexpression of Cas increases the amount of GTP-bound Rap1 like Chat. Co-expression of Cas with Chat enhanced the Rap1 activation (Chat+Cas), but the C-terminal portion of Cas inhibited it (Chat+Cas-C-half). (B) Rap1 activation by Chat, Cas or Crk overexpression. Overexpression of Crk showed the strongest Rap1 activation. Enhanced Rap1 activation by co-expression of Chat and Cas with Crk was hard to detect because of the robustness of the Crk-induced Rap1 activation.

 


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  		Fig. 4. Deletion effect of Cas domain structure on Cas-induced Rap1 activation. (A) Schematic representation of Cas variants used for the evaluation of Rap1 activation competence. (B) Overexpression effect of Cas and its deletion mutants on Rap1 activity was examined as in Fig. 1. Cas overexpression increases the amount of GTP-bound Rap1 compared with control pCAX transfectant. Cas {Delta}SD failed to activate Rap1, whereas Cas {Delta}SCB substantially retained the activity. (C) Tyrosine phosphorylation and Crk interaction of Cas mutants. 293T cells were transfected with a Cas derivative expression plasmid together with Crk. After immunoprecipitation with anti-HA antibody, the tyrosine phosphorylation levels of Cas proteins and co-immunoprecipitated Crk in the immunoprecipitates were analyzed.

 


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  		Fig. 5. Suppression of Myr-Chat-mediated Rap1 activation by dominant-negative mutants of Cas, Crk or C3G. 293T cells were co-transfected with Myr-Chat and Cas, Crk or C3G mutant expression plasmid. After 30 hours, the Rap1 activation level of these transfected cells was examined. Co-expression of Cas {Delta}SD (Myr+{Delta}SD), Crk W169L (Myr+W169L) or C3G dCD (Myr+dCD) effectively suppressed the Rap1 activation induced by Myr-Chat.

 


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  		Fig. 6. Chat is associated with the Cas-Crk signaling complex. The association of Chat with Cas or Crk was examined by a co-immunoprecipitation assay. 293T cells were transfected with (+) or without (-) expression plasmids for Chat, Cas or Crk. Transfected cell lysates were immunoprecipitated with anti-Cas or anti-Crk antibodies; co-immunoprecipitated Chat was detected by an anti-Chat immunoblot. The expression level of Chat, Cas and Crk was analyzed by total lysate immunoblot of each protein. The different mobilities of endogenous Crk (Crk) and Myc-tagged Crk (Myc-Crk) are shown in the right side of the panel. The amount of Chat co-immunoprecipitated with Cas was not affected by Crk expression level. In Chat and Crk co-expressing cells, the amount of Chat co-immunoprecipitated with Crk was not increased. When Cas was expressed together with Chat and Crk, a marked increase of Chat in Crk immunoprecipitate was observed.

 


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  		Fig. 7. Membrane targeting of Chat induces cell periphery spreading and branching. NIH 3T3 cells were transfected with an expression plasmid for Chat (A,B) or Myr-Chat (C,D,E). Intracellular localization of expressed proteins was detected by anti-FLAG tag immunofluorescence (A,C,E). Higher magnification of C is shown in E. Cell shapes were visualized with phalloidin staining of the actin filament (B,D). Chat was detected mainly in the cytoplasm (A). Myr-Chat was localized at the plasma membrane and intracellular vesicle like spots (C,E). Myr-Chat but not Chat expression induced spreading of cell periphery, which was frequently accompanied by branched cell morphology (B,D). Bars, 10 µm.

 


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  		Fig. 8. Myr-Chat-expressing cells show a highly enlarged cell area. NIH 3T3 cells were transfected with indicated pIRES-EGFP-based expression plasmids. Phase-contrast images (left column) and fluorescence images (middle column) of EGFP-positive cells are shown. The cell area of transfected cells was evaluated from the fluorescence-emitting region of EGFP-positive cells and represented as histograms on the right column (EGFP, n=52, MED=1335.6 µm2; Chat, n=44, MED=1266.3 µm2; Myr-Chat, n=39, MED=1748.0 µm2; Myr-{Delta}CT, n=45, MED=1209.5 µm2). Membrane-targeted Myr-Chat expression induced cell elongation, which was frequently accompanied by a branched morphology. These cells also showed increased cell area value compared with control pIRES-EGFP-transfectants. Wild-type Chat-transfectants did not show any apparent morphological alterations. Myr-Chat-{Delta}CT also did not show the cell area enlarging activity. Bar, 10 µm.

 


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  		Fig. 9. The morphological effect of Myr-Chat is suppressed by co-expression of dominant-negative mutant of Cas, Crk, C3G or Rap1. NIH 3T3 cells were transfected with pIRES-EGFP (EGFP) or pIRES-EGFP-Myr-Chat (Myr-Chat) together with a plasmid expressing a mutant of Cas (Myr+Cas {Delta}SD), Crk (Myr+Crk W169L), C3G (Myr+C3G dCD) or Rap1 (Myr+N17 Rap1). Analyses of cell morphology and cell area of transfected cells were performed as in Fig. 8 (EGFP, n=66, MED=1118.3 µm2; Myr-Chat, n=57, MED=1699.7 µm2; Myr+Cas {Delta}SD, n=57, MED=1089.4 µm2; Myr+Crk W169L, n=58, MED=1068.6 µm2; Myr+C3G dCD, n=57, MED=864.9 µm2; Myr+N17 Rap1, n=65, MED=1063.5 µm2). Expression of Myr-Chat increased the population of cells with protrusions and branched shape, which caused a cell area value increase. Co-expression of Cas {Delta}SD, Crk W169L, C3G dCD or N17 Rap1 suppressed the cell-shape-converting effect of Myr-Chat. Bar, 10 µm.

 


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  		Fig. 10. Myr-Chat expression enhances the cell adhesion on fibronectin. 293T cells were transfected with a control pCAX or indicated Chat variant plasmid together with pCAX-EGFP. Cell adhesion on 96-well culture plates coated with 0.2 µg/ml fibronectin was measured as described in the Materials and Methods. Mean values obtained from the average of six independent wells are shown with s.d. The asterisk indicates the value with a statistically significant increase (P<0.01) over control cells determined by Student's t-test. Control and Chat-transfected cells adhered to the fibronectin matrix to a similar extent. Myr-Chat-expressing cells showed a 1.5 times increase of adhesion compared with control cells. Expression of Myr-{Delta}CT did not enhance the adhesion.

 

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© The Company of Biologists Ltd 2002