A Pyk2-Vav1 complex is recruited to β3-adhesion sites to initiate Rho activation

Integrin alpha v beta 3-mediated adhesion of haemopoietic cells to vitronectin results in beta 3 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 alpha v beta 3 during leucocyte adhesion to vitronectin. In the present Study, we have identified the nonreceptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with alpha v beta 3. The association of Pyk2 and Vav1 with beta 3 relies on the presence of Tyr(747) in beta 3, the primary site of beta 3 phosphorylation. However, association of Pyk2 with Vav1 is independent of beta 3 tyrosine phosphorylation. Formation of a Pyk2-Vav1 complex occurs upon cell adhesion and Pro(717) of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 it? 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 beta 3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of beta 3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.