CrkII regulates focal adhesion kinase activation by making a complex with Crk-associated substrate, p130Cas.
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.
Iwahara
Toshinori
T
Laboratory of Molecular Oncology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.
Akagi
Tsuyoshi
T
Fujitsuka
Yuki
Y
Hanafusa
Hidesaburo
H
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
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