Pyk 2 FAKs, any two FAKs.

Recent data suggest a novel and significant role for the protein tyrosine kinase Pyk2/CAK /RAFTK in cytoskeletal remodeling, proliferation and motility of adherent cells, such as epithelial cells and fibroblasts, possibly putting this kinase in direct competition with its close cousin, the focal adhesion kinase, FAK. Within a three-month period in late 1995, separate groups identified the same novel protein tyrosine kinase (PTK). Lev et al. (1995) identified a “proline-rich tyrosine kinase,” Pyk2, as a PTK highly expressed in the nervous system, whose activity, induced by PKC and high intracellular calcium, modulates ion channel function and MAP kinase activity. Secondly, Sasaki et al. (1995) cloned cell adhesion kinase beta, CAK , a focal adhesion kinase (FAK) homologue that is possibly involved in signal transduction mediated by cell–cell interactions. Lastly, Avraham et al. (1995) isolated a “related adhesion focal tyrosine kinase” RAFTK, enriched in brain and induced by thrombin treatment of megakaryocytes. Unlike FAK, which is expressed throughout the body and in all cell types, Pyk2/CAK / RAFTK (referred to hereafter as Pyk2) is expressed at higher levels in the brain, hematopoietic cells and osteoblasts, and to a lesser extent in certain epithelial cells, smooth muscle cells and fibroblasts (Avraham et al., 2000) (Fig. 1 ). Previous studies have localized Pyk2 to perinuclear regions in fibroblasts using indirect immunofluorescence with polyclonal antibodies. However, this finding was probably influenced by the antibodies used. On one hand, cytoplasmic Pyk2 staining was detected using (i) a monoclonal antibody (clone 11) developed by Transduction Laboratories (Sieg et al., 1998), (ii) a rabbit polyclonal raised against the C-terminal 250 amino acid (a.a.) of Pyk2 (Zheng et al., 1998), (iii) a polyclonal antibody raised to Pyk2 a.a. 587–988 (Xiong and Parsons, 1997), (iv) an epitope-specific monoclonal to detect myc-tagged Pyk2 (Klingbeil et al., 2001), or (v) an epitope-specific monoclonal to detect HA-tagged Pyk2 (Zhao et al., 2000). In contrast, staining of Pyk2 was detected in focal adhesions using (i) an epitope-specific monoclonal to detect KT3-tagged Pyk2 (Schaller and Sasaki, 1997), (ii) a rabbit polyclonal antibody specific for Pyk2 (i.e. no cross-reactivity to FAK) from Upstate Biotechnology (Du et al., 2001), or (iii) rabbit polyclonal antibodies specific for the Pyk2 Y402 and Y881 phospho-tyrosine domains (Nakamura et al., 2001). To complicate matters, Schaller and Sasaki (1997) showed that a Pyk2 C-terminal domain stains in focal adhesions, whereas full-length Pyk2 stains throughout the cell. There is also reason to suspect that antibody sensitivity plays a role, because Pyk2 focal adhesion staining is especially appreciated under conditions of overexpression (Du et al., 2001). If Pyk2 localizes to focal adhesions, does it play a role in cytoskeletal remodeling or cellular functions involving focal adhesion turnover, such as motility, and if so, how does Pyk2 affect FAK function? A previous study by Schaller and Sasaki (1997) using chicken embryo fibroblasts, as well as a recent paper by Du et al. (2001) using Swiss 3T3, 3Y1 and 10T1/2 fibroblasts, and MDCK epithelial cells, both demonstrate that Pyk2 overexpression induces cell rounding, enlargement of focal adhesion complexes into podosome-like structures and marginalization of F-actin to the cell periphery reminiscent of FAK-deficient mouse embryo fibroblasts. In contrast, FAK overexpression did not grossly reorganize the actin-based cytoskeleton in these cell types, but significantly, Pyk2 catalytic activity was required for cytoskeletal reorganization. Thus, the epithelioid morphology of FAK / mouse embryo fibroblasts (MEF) may actually be due to their 2to 3-fold higher levels of Pyk2 (Sieg et al., 1998) rather than the lack of FAK, as was previously assumed. In agreement with this, * Corresponding author. Tel.: +1-716-845-7681; fax: +1-716-845-1698 E-mail address: irwin.gelman@roswellpark.org (I.H. Gelman). Cell Biology International 27 (2003) 507–510 Cell Biology International