title: CrkII regulates focal adhesion kinase activation by making a complex with Crk-associated substrate, p130Cas.
abstract: CrkII is an adaptor protein possessing oncogenic potential despite the lack of an enzymatic domain. We investigated here the physiological functions of CrkII by studying its ability to induce anchorage-independent cell growth. We found that inhibition or null mutation of focal adhesion kinase (FAK) blocked the anchorage-independent growth induced by CrkII overexpression, indicating that FAK is a critical determinant of the transforming activity of CrkII. CrkII overexpression enhanced the autophosphorylation of FAK at Tyr-397 and tyrosine phosphorylation of p130(Cas) (Crk-associated substrate, Cas) upon stimulation of integrin by fibronectin. Moreover, the constitutive phosphorylation of FAK and Cas was observed in CrkII-overexpressing cells, even when they were in the suspended condition, consistent with the ability of CrkII to induce anchorage-independent growth. Using Cas-deficient cells, we showed Cas function to be essential for both the CrkII-induced phosphorylation of FAK (Tyr-397) and anchorage-independent cell growth. The CrkII-induced FAK autophosphorylation depended upon CrkII-Cas complex formation. Furthermore, we showed that CrkII knockdown resulted in defects in integrin-mediated events, such as cell spreading, haptotactic migration, and FAK autophosphorylation. The integrin-mediated FAK autophosphorylation was also reduced in Cas-deficient cells. These results suggest that the CrkII-Cas complex functions in integrin-mediated FAK activation signaling. Our findings show the importance of CrkII in integrin-mediated events, acting upstream of FAK to affect the activation of this kinase, which appears to have a central role in this pathway.
authors.affiliation: Laboratory of Molecular Oncology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.
authors.forename: Toshinori
authors.initials: T
authors.lastname: Iwahara
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Akagi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fujitsuka
authors.forename: Hidesaburo
authors.initials: H
authors.lastname: Hanafusa
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D050719:Crk-Associated Substrate Protein; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015870:Gene Expression; D006801:Humans; D016023:Integrins / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D050717:Retinoblastoma-Like Protein p130
chemical_list: C493841:BCAR1 protein, human; C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D016023:Integrins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D050717:Retinoblastoma-Like Protein p130; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse