Activation of Protein-tyrosine Kinase Pyk2 Is Downstream of Syk in FcεRI Signaling

Abstract

Aggregation of the Fc⑀RI, a member of the immune receptor family, induces the activation of proteintyrosine kinases and results in tyrosine phosphorylation of proteins that are involved in downstream signaling pathways. Here we report that Pyk2, another member of the focal adhesion kinase family, was present in the RBL-2H3 mast cell line and was rapidly tyrosinephosphorylated and activated after Fc⑀RI aggregation. Tyrosine phosphorylation of Pyk2 was also induced by the calcium ionophore A23187, by phorbol myristate acetate, or by stimulation of G-protein-coupled receptors. Adherence of cells to fibronectin dramatically enhanced the induced tyrosine phosphorylation of Pyk2. Although Src family kinases are activated by Fc⑀RI stimulation and tyrosine-phosphorylate the receptor subunits, the activation and tyrosine phosphorylation of Pyk2 were downstream of Syk. In contrast, tyrosine phosphorylation of Pyk2 by stimulation of G-protein-coupled receptors was independent of Syk. Therefore, the Fc⑀RI-induced tyrosine phosphorylation of Pyk2 is downstream of Syk and may play a role in cell secretion.The aggregation of the Fc⑀RI 1 on basophils or mast cells initiates a cascade of biochemical events that results in degranulation and the release of inflammatory mediators (1, 2). Tyrosine phosphorylation of proteins plays a critical role in this signal transduction pathway (3-8). Among the signal transduction molecules that are tyrosine-phosphorylated are the ␤ and ␥ subunits of the receptor, Lyn, Syk, phospholipase C-␥1 and -␥2, Vav, Btk, and the focal adhesion kinase FAK (9 -15). One of the earliest events after aggregation of Fc⑀RI is the activation of protein-tyrosine kinases, probably Lyn or another Src family kinase, that results in the tyrosine phosphorylation of the ␤ and ␥ subunits of the receptor (9, 16). The proteintyrosine kinase Syk is then recruited by the tyrosine-phosphorylated receptor and is critical for the downstream activation signals (11,(17)(18)(19). For example, Fc⑀RI aggregation in a Sykdeficient mast cell line does not mobilize Ca 2ϩ from intracellular and extracellular sources and fails to propagate downstream signaling events (20).Previously we observed that Fc⑀RI aggregation results in the tyrosine phosphorylation of ϳ115-kDa proteins in the rat basophilic leukemia RBL-2H3 mast cell line (21,22). Two of these proteins were identified as FAK and the cell surface adhesion molecule CD31 (15,23). Recently Pyk2 was identified as another member of the FAK family of protein-tyrosine kinases (24 -26). Pyk2 (also called RAFTK for related adhesion focal tyrosine kinase, CAK␤ for cell adhesion kinase ␤, CADTK and FAK2) is a cytoplasmic protein-tyrosine kinase that, like FAK, lacks a transmembrane region, myristoylation sites, and Src homology 2 and 3 domains. Both FAK and Pyk2 have a central kinase region flanked by large N-terminal and C-terminal domains. Pyk2 is expressed in neuronal cells, CD34ϩ bone marrow cells, primary bone marrow megakaryocytes, platelets, and T and B cells (24,26,27).

Cutting Edge: Functional Role for Proline-Rich Tyrosine Kinase 2 in NK Cell-Mediated Natural Cytotoxicity

“…At present, it is unclear whether there is any functional interdependence between Pyk2 and Syk upon NK cell interaction with target cells. It has been recently reported that Syk activation is central to the generation of both natural cytotoxicity and ADCC (11) and that Pyk2 activation may occur through Syk-dependent and independent pathways (7). Based on this observation, our data suggest that either Pyk2 activation does not require Syk and Pyk2 may cooperate with this PTK to fully activate natural cytotoxicity or Pyk2 is upstream to Syk.…”

“…Pyk2/related adhesion focal tyrosine kinase/cell adhesion kinase-␤ is expressed in different cell types including brain cells, fibroblasts, and hemopoietic cells (1)(2)(3)(4)(5)(6)(7). In hemopoietic cells, Pyk2 and its alternatively spliced isoform, Pyk2-H, are activated by cytokines, chemokines, and through a number of receptors including multichain immune recognition receptors and integrins (4 -8).…”

Nuclear Translocation of Cell Adhesion Kinase .BETA./Proline-rich Tyrosine Kinase 2.

“…CAKβ is activated by osmotic stress and in response to stimulation of G-protein-coupled receptors and receptor tyrosine-kinases (Dikic et al, 1996). It was also shown that CAKβ/PYK2 is activated by stimulation of the T-cell receptor (Qian et al, 1997), interleukin-2 receptor (Miyazaki et al, 1998), interferon-γ receptor (Takaoka et al, 1999), and Fcε receptor I (Okazaki et al, 1997). CAKβ has binding sites for p130 Cas (Cas), Graf, Hic-5, and paxillin and these proteins are tyrosine-phosphorylated upon CAKβ activation (Matsuya et al, 1998;Astier et al, 1997;Salgia et al, 1996;Hildebrand et al, 1996).…”

Lipopolysaccharide Induces Actin Reorganization and Tyrosine Phosphorylation of Pyk2 and Paxillin in Monocytes and Macrophages

“…Pyk2 expression is more limited than FAK, and has been detected in epithelial cells, neuronal cells, T and B cells, megakaryocytes, platelets, mast cells, and monocytes and macrophages (26,30,31), whereas Pyk2-H is only expressed in hemopoietic cells (28,29). The stimulation of many types of cell surface receptors results in the tyrosine phosphorylation of Pyk2, including integrins, cytokines, and immune receptors (30,(32)(33)(34); and diverse stimuli such as membrane depolarization, stress stimuli, angiotensin, and PMA have all been shown to induce Pyk2 tyrosine phosphorylation, resulting in the activation of its kinase activity (26,31).…”