A Pyk2-Vav1 complex is recruited to beta3-adhesion sites to initiate Rho activation.
Integrin alphavbeta3-mediated adhesion of haemopoietic cells to vitronectin results in beta3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with alphavbeta3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with alphavbeta3. The association of Pyk2 and Vav1 with beta3 relies on the presence of Tyr747 in beta3, the primary site of beta3 phosphorylation. However, association of Pyk2 with Vav1 is independent of beta3 tyrosine phosphorylation. Formation of a Pyk2-Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)-Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and beta3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of beta3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.
Gao
Chunlei
C
Department of Cell and Development Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
Blystone
Scott D
SD
D001665:Binding Sites; D002448:Cell Adhesion; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D039661:Integrin beta3 / Q000378:metabolism*; D020014:K562 Cells; D010766:Phosphorylation; D021381:Protein Transport; D051567:Proto-Oncogene Proteins c-vav / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D039302:Integrin alphaVbeta3; D039661:Integrin beta3; D051567:Proto-Oncogene Proteins c-vav; C496618:VAV1 protein, human; D051418:Focal Adhesion Kinase 2; D020741:rho GTP-Binding Proteins