Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites.
Rac1, a member of the Rho family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of Rac1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged Rac1, we demonstrate that EGFP-Rac1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-Rac1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-Rac1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced Rac1 activation by measuring the amounts of GTP-bound Rac1 based on its specific binding to the Cdc42/Rac1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced Rac1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of Rac1, and EGFP-Rac1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. Rac1 activation was also confirmed by measuring the amounts of coimmunoprecipitated Rac1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that Rac1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase.
Nakagawa
M
M
Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.
Fukata
M
M
Yamaga
M
M
Itoh
N
N
Kaibuchi
K
K
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015820:Cadherins / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D022001:Focal Adhesions / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D007365:Intercellular Junctions / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011233:Precipitin Tests; D015398:Signal Transduction / Q000502:physiology; D000077191:Wortmannin; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins
D000730:Androstadienes; D000911:Antibodies, Monoclonal; D015820:Cadherins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; C101203:IQ motif containing GTPase activating protein 1; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D020703:ras GTPase-Activating Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D000077191:Wortmannin