CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase

The proline-rich tyrosine kinase 2, Pyk2, is a focal adhesion related kinase expressed in T cells that is tyrosine phosphorylated and activated by integrin, chemokine or T cell receptor stimulation. Ligation of the cell adhesion molecule CD44 also induces Pyk2 phosphorylation and T cell spreading, and this is negatively regulated by the protein tyrosine phosphatase CD45. Here, we identify the activation requirements for Pyk2 and demonstrate its requirement for CD44-mediated elongated T cell spreading. Upon CD44-mediated cell spreading, Pyk2 was recruited to CD44 clusters in both CD45⁺ and CD45⁻ T cells, yet was more strongly phosphorylated in T cells lacking CD45. In these cells, Pyk2 phosphorylation was dependent on Src family kinase activity and required actin polymerisation, phosphatidylinositol-3 kinase and phospholipase C activity as well as extracellular calcium. Inhibition of any of these events prevented Pyk2 phosphorylation and T cell spreading. Transfection of a truncated form of Pyk2 lacking the kinase domain, PRNK, inhibited CD44-mediated cell spreading, demonstrating an important role for Pyk2. However, inhibition of microtubule turnover by Taxol prevented elongated T cell spreading but did not affect Pyk2 phosphorylation, indicating that microtubule reorganisation is downstream, or independent, of Pyk2 phosphorylation. Together this demonstrates that multiple factors are required for CD44-induced Pyk2 activation, which plays a critical role in CD44-mediated elongated T cell spreading.     

Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor

Wounding usually causes considerable cell damage, and released ATP promotes migration of nearby epithelium. ATP binds to purinergic receptors on the cell surface and induces transactivation of the EGF receptor through signaling by the Src family kinases (SFKs). Here we tested whether ATP activates these kinases through Pyk2, a member of the focal adhesion kinase family. Pyk2 was rapidly and potently activated by treating corneal epithelial cells with ATP, and physical interaction of Pyk2 with the SFKs was enhanced. Disruption of Pyk2 signaling either by siRNA or by expression of a dominant-negative mutant led to inhibition of ATP-induced activation of the SFKs and the EGF receptor. Inhibiting Pyk2 activity also blocked ATP stimulation of healing of wounds in epithelial cell sheets. These data suggest that ATP stimulates sequential activation of Pyk2, SFKs, and the EGF receptor to induce cell migration.