Abstract
Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.
Highlights
Upon cell adhesion to extracellular matrix, the integrin-family of transmembrane receptors are clustered at sites termed focal adhesions and activate various intracellular signaling pathways
While this initial phosphorylation of Proline-rich tyrosine kinase 2 (Pyk2) by Src is independent of Pyk2 kinase activity, our results suggest that subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation
We observed that fibronectin-mediated Pyk2 phosphorylation was significantly reduced at both the Y402 and Y579 sites by these inhibitors (Fig 1B)
Summary
Upon cell adhesion to extracellular matrix, the integrin-family of transmembrane receptors are clustered at sites termed focal adhesions and activate various intracellular signaling pathways. Focal adhesion kinase (FAK) family members are important downstream mediators of integrin signaling in events such as cell proliferation, survival, motility and invasion, and are considered as plausible drug targets in various disease processes, such as inflammation and cancer [1,2]. Proline-rich tyrosine kinase 2 (Pyk2), known as RAFTK and CAK-β, is a non-receptor tyrosine kinase that shares related structure and sequence similarity with FAK [1,3,4]. Both of these kinases comprise an N-terminal FERM domain, a central kinase domain and a focal adhesion targeting (FAT) domain towards the C-terminus.
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