Pyk2 specific features

As mentioned before, Pyk2 and FAK show a similar architecture of FERM, kinase and FAT domain and share common features in regard to auto-inhibition and activation (Fig. 1.6). However, also important differences within their structure exist that are responsible for differences in FAK versus Pyk2 signaling. Pyk2 was shown to be activated upon stimuli that increase intracellular Ca²⁺-levels and its FERM

20 domain is suggested to directly bind Ca²⁺-bound calmodulin (Fig. 1.6) (Lev et al., 1995; Kohno et al., 2008; Xie et al., 2008). This binding is specific for the Pyk2-FERM domain and was shown to induce dimerization or even oligomerization of Pyk2 resulting in trans activation at the auto-phosphorylation site Y402 (Kohno et al., 2008; Riggs et al., 2011). Similar to FAK, also Pyk2 is able to bind Src family kinases (SFK) upon auto-phosphorylation at Y402 resulting in the phosphorylation of additional tyrosine residues (Y579/580) and full activation of the kinase (Sasaki et al., 1995; Dikic et al., 1996; Li et al., 1996). The Pyk2 kinase domain shows classical conformational features as seen for FAK and other protein tyrosine kinases including the bi-lobed folding and structural flexibility of the activation loop (Han et al., 2009). Similar to FAK, Pyk2 also harbors proline rich regions (PRR) between the kinase and the FAT domain enabling interactions with adaptor proteins like p130cas (Nojima et al., 1995; Astier et al., 1997). Also the FAT domain of Pyk2 shows a quite high sequence similarity (~40%) to FAK-FAT and exhibits a similar domain arrangement (Mitra et al., 2005; Lulo et al., 2009). Although both FAT domains were shown to interact with Paxillin, the FAT domain of Pyk2 does not promote such strong focal adhesion localization of the full-length protein as it can be observed for FAK (Turner et al., 1993; Tachibana et al., 1995; Salgia et al., 1996; Hoellerer et al., 2003). This might be due to different binding partners and/or variations in the binding affinity. In contrast to the FAK-FAT domain Pyk2-FAT does not bind to the FA protein Talin (Chen et al., 1995; Zheng et al., 1998). Although Talin is not the main determinant of FAK FA localization this may at least partially contribute to the weak FA localization of Pyk2. In addition to Paxillin, Pyk2-FAT is reported to bind the Paxillin-related protein Hic-5 that localizes to FAs but can also enter the nucleus and may be responsible for nuclear shuttling of Pyk2 (Matsuya et al., 1998; Aoto et al., 2002). The interaction between Pyk2-FAT and another Paxillin family member (Leupaxin) might represent a relevant, cell-type specific interaction as both proteins are expressed in hematopoietic cells (Lipsky et al., 1998; Vanarotti et al., 2016). By comparing the interactions of Pyk2 and Paxillin or Leupaxin, it could be shown that Pyk2 preferably binds Leupaxin, which suggests that Leupaxin is the native Pyk2 binding partner (Vanarotti et al., 2016). Similar to FRNK, there exists also a Pyk2 variant consisting of only the C-terminus without any kinase function, called PRNK (Pyk2-related non-kinase). PRNK is also mainly expressed in hematopoietic cells and shows a better focal adhesion localization compared to full-length Pyk2 (Xiong

21 et al. 1998). As seen for FRNK and FAK, also the overexpression of PRNK is able to inhibit Pyk2-dependent processes like cell migration (Watson et al., 2001; Zhu et al., 2008; Wang et al., 2010). PRNK could therefore enable a cell type-specific control of cellular processes depending on its expression pattern.

Figure 1.4: Pyk2 has adopted the domain organization of FAK. The structures of the isolated Pyk2 FERM (green) and kinase (cyan) domains are shown. Pyk2 key residue F599 (blue), corresponding to FAK Y596, is highlighted. Reported Ca²⁺/Calmodulin binding motifs are mapped onto the FERM and kinase domains (orange) (taken from Walkiewicz et al., 2015).

Pyk2 was shown to be involved in a variety of cellular processes. Knockdown or knockout of Pyk2 in macrophages strongly impaired cell migration (Duong and Rodan, 2000; Okigaki et al., 2003). This might be due to defects in the contractile activity within lamellipodia through reduced Rho GTPase activity (Okigaki et al., 2003). Pyk2 was not only shown to be important for the mobility of blood cells, but is also crucial for migration of astrocytes during wound healing (Giralt et al., 2016).

The importance of Pyk2 for cell migration connects this kinase also to Integrin signaling. Indeed, stimulation of different Integrin heterodimers, like αVβ3 or αMβ2

(CR3) leads to the activation and recruitment of Pyk2 (Duong and Rodan, 2000;

Butler et al., 2005; Gao et al., 2009; Wang et al., 2010). In addition to migration, Pyk2 was also shown to be involved in the Integrin-dependent uptake of pathogenic bacteria. Infection with Yersinia species that express the virulence factor YadA,

Pyk2

22 which can indirectly bind to β1 Integrins leads to the activation of Pyk2 and FAK (Bruce-Staskal et al., 2002; Eitel and Dersch, 2002; Hudson et al., 2005; Owen et al., 2007). Furthermore, Pyk2 was uncovered to be important for the generation of a respiratory burst after Salmonella and Listeria infections, but not for the intracellular killing of these pathogens (Han et al., 2003). Contrastingly, another study connects Pyk2 to the degranulation response upon Staphylococcus aureus infection, but shows no role of Pyk2 for the respiratory burst (Kamen et al., 2011).

Increased Pyk2 activity and expression in blood cells from patients with the autoimmune disease systemic lupus erythematosus (SLE) further demonstrates a role of Pyk2 in inflammatory immune responses (Wang et al., 2009).

In addition to its conventional role, Pyk2 can also partially take over FAK specific functions and is overexpressed in cells lacking FAK (Lim et al., 2008). For example, FAK deletion or inhibition in endothelial cells leads to a switch to Pyk2-dependent signaling that can compensate for FAK-loss during angiogenesis (Weis et al., 2008).

Furthermore, targeting the Pyk2 FERM/kinase domains to focal adhesions via fusion to the FAK C-terminus restores fibronectin-induced migration in FAK knockout fibroblasts (Klingbeil and Hauck et al., 2001). This shows that Pyk2 is able to act in part like FAK and both kinases need to be controlled differentially to execute their characteristic functions.