FAK domains and activation

FAK is an essential protein tyrosine kinase in multicellular organisms as genetic deletion of FAK results in an early embryonic lethal phenotype (Ilic et al. 1995).

FAK is an ubiquitously expressed 125 kDa non-receptor protein tyrosine kinase (PTK) and is highly conserved across species. Proline-rich tyrosine kinase 2 (Pyk2) is the second member of the family of FAK kinases. Pyk2 shares some structural and functional similarities with FAK, however, they display distinct differences and are clearly not redundant (Schaller 2010). Structurally, FAK comprises three domains and localizes at sites of clustered integrins, so called focal adhesions. At the N-terminus a band 4.1, Ezrin, Radixin, Moesin-domain (FERM-domain) is found, followed by the central kinase domain and a C-terminal focal adhesion targeting (FAT) domain (Fig. 1.4).

Between the FERM and the kinase domain one proline-rich region (PRR) is lo-cated, two additional PRRs are found between the kinase and the FAT domain, that function as binding sites for Src-homology (SH) 3 domain containing pro-teins. The FAT domain harbors four amphipathic α-helices which tightly pack together by hydrophobic interactions into an antiparallel four-helix bundle (Hayashi et al. 2002). It contains binding sites for the focal adhesion proteins Talin and Paxillin (Chen et al. 1995; Tachibana et al. 1995). While binding to Talin is not essential for the initial recruitment of FAK (Lawson et al. 2012), binding to Paxillin seems to mediate focal adhesion targeting of FAK. A striking feature of the FAT domain are two hydrophobic patches on the surface flanked by basic residues at the interface of α-helices 1 and 4 and on the opposite site of the molecule at the interface of α-helices 2 and 3. These hydrophobic patch-es bind to the hydrophobic surface of the second and fourth leucine-rich domain (LD) motifs from Paxillin (Hayashi et al. 2002; Bertolucci et al. 2005). Point mu-tations within the Paxillin-binding motifs of the FAK FAT domain disrupt FAK association emphasizing the role of this specific interaction for FAK localization at focal adhesions (Scheswohl et al. 2008). Structural analyses suggest a dy-namic nature of FAT adopting either the four-helix bundle or an alternative con-formation allowing phosphorylation of Tyr925 (Hall et al. 2011). Tyr925 phos-phorylation seems to induce the dislocation of FAK from focal adhesions (Katz

Fig. 1.4: Scheme of functional domains of FAK and FAK interaction partners (taken from (Chatzizacharias et al. 2008)).

The FERM domain is trilobed comprising the F1, F2 and F3 subdomains. FERM domains often act as linkers between the cytoskeleton and the plasma mem-brane and are typically located at the N-terminus (Chishti et al. 1998). In the inactive state the FERM domain is bound to the FAK kinase domain and pre-vents access to the catalytic cleft and autophosphorylation at Tyr397 (autoinhib-ited conformation) (Cooper et al. 2003; Lietha et al. 2007). Furthermore, Tyr576 and Tyr577 in the activation loop are sequestered and unavailable as sub-strates for Src (Lietha et al. 2007). Deletion of the FERM domain is associated with elevated catalytic activity and/or tyrosine phosphorylation indicating the negative regulatory function (Schlaepfer and Hunter 1996; Toutant et al. 2002;

Jacamo and Rozengurt 2005). Cell matrix contact, mechanic stress or growth factors cause FAK activation (Schlaepfer and Hunter 1998; Schaller et al. 1999;

Mitra et al. 2005). Stimulation of fibroblasts promotes FAK binding via its FERM domain to the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors accompanied by FAK activation (Sieg et al. 2000). The FERM F2 subdomain contains a basic patch with the sequence KAKTLRK that is important for the activation of FAK following cell adhesion and stimulation with hepatocyte growth factor (HGF) by direct binding to activated Met or inter-action with phosphoinositides (Chen and Chen 2006; Cai et al. 2008; Chen et al. 2010). Activation of FAK involves conformational changes to release the in-tramolecular inhibitory association between the FERM domain and the kinase domain (Lietha et al. 2007; Cai et al. 2008) accompanied by Tyr397 phosphory-lation in cis or also in trans by other cellular tyrosine kinases. Phosphorylated Tyr397 serves as a docking site for SH2 domain containing proteins in particular Src. Src phosphorylates Tyr576 and Tyr577 within the activation loop to gain full catalytic activity of FAK (Calalb et al. 1995) (Fig. 1.5).

Fig. 1.5: The cycle of FAK activation/inactivation. The autoinhibited conformation of FAK is shown with the FERM and kinase domains forming a direct interaction blocking access of ATP and substrate to the active site. (1) The first step of activation requires ligand binding releasing autoinhibition. (2) Autophosphorylation of FAK creates an SH2 binding site at Tyr397. Phos-phorylation of this site could further destabilize the autoinhibitory conformation through disrup-tion of the interacdisrup-tion between the linker and the F1 subdomain, although this remains to be determined. (3) The Tyr397 SH2 binding site can act as a scaffold to recruit various signaling molecules into complex. (4) This same site is the binding site for Src, which is responsible for phosphorylation of the activation loop resulting in maximal activation of FAK catalytic activity.

The FAK/Src complex represents the most active enzyme complex in the cycle. (5) When Src is released, FAK retains maximal activity due to phosphorylation of the activation loop. (6) After release of Src, the fully active FAK kinase may scaffold other SH2 domain-containing proteins.

(7) Return to the autoinhibited conformation requires phosphatase activity. The details of this step have yet to be elucidated (taken from (Hall et al. 2011)).

It is assumed that phosphorylation of the activation loop blocks the FERM-kinase domain interaction (Hall et al. 2011). The activated FAK/Src complex binds to and phosphorylates substrates like Paxillin (Schaller and Parsons 1995) and p130Cas (Tachibana et al. 1997) and plays a central role in the

RhoGTPases-mediated reorganization of the actin cytoskeleton (Mitra et al.

2005) (compare also Fig. 1.4).

1.2.2 FAK as a central regulator of focal adhesion dynamics and