Proline-Rich Tyrosine Kinase-2 Activation by β1 Integrin Fibronectin Receptor Cross-Linking and Association with Paxillin in Human Natural Killer Cells 1

Recent evidence indicates that integrin ligation results in activation of focal adhesion kinase (pp125^FAK), the prototype of a new subfamily of nonreceptor protein tyrosine kinase (PTK), including FAKB and the proline-rich tyrosine kinase 2 (PYK-2), also termed cell adhesion kinase-β or related adhesion focal tyrosine kinase. We have previously shown that cross-linking of α₄β₁ and α₅β₁ fibronectin receptors on human NK cells stimulates tyrosine phosphorylation of two proteins migrating at 105 and 115 kDa. Here we report that cross-linking of β₁ integrins on human NK cells stimulates tyrosine phosphorylation and PTK activity of PYK-2. PYK-2 tyrosine phosphorylation was maximal at 1 min and started to decline 20 min after stimulation. Engagement of α₄β₁ and α₅β₁ either with specific mAbs or after cell adhesion to fibronectin or its 120- and 40-kDa fragments also triggered PYK-2 tyrosine phosphorylation. Stimulation of PYK-2 tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by EGTA, indicating that PYK-2 tyrosine phosphorylation is PTK, but not calcium, dependent. We also demonstrate that PYK-2 is constitutively associated with paxillin, which undergoes tyrosine phosphorylation with the same kinetics of PYK-2 upon β₁ integrin ligation.