Focal Adhesion kinase (FAK)

1.2.1. Activation of FAK by integrins

The cloning and characterization of FAK were reported in 1992 (90; 91). FAK is a non-receptor tyrosine kinase, which consists of a central tyrosine kinase (catalytic) domain flanked by large N- and C-terminal non-catalytic domains (91; 95). The N-terminal region contains a FERM domain that is thought to mediate interactions of FAK with proteins associated with the plasma membrane. The C-terminal domain of FAK, designated “FAT”, for Focal Adhesion Targeting, is essential for localizing FAK to integrin-adhesion sites (92; 95). Finally, two proline-rich motifs (PR1 and PR2) are located between the kinase and the FAT domains (Fig. 6). These motifs mediate interactions with Src-homology 3 (SH3)-domain-containing proteins. These functional and structural characteristics of FAK have led to its definition as a “scaffolding protein with kinase activity”.

FAK is localized in focal adhesions (90; 91), and is widely expressed in different tissues (90). The first indication that FAK is a signaling protein was provided by the demonstration that it is phosphorylated on tyrosine residues when cells are plated on the ECM molecule fibronectin (90; 96; 97). It was subsequently reported that other ECM proteins (laminin, collagen IV and vitronectin) also induce tyrosine phosphorylation of FAK (89; 98). In addition, the overall phenotype of the FAK knock-out mouse is similar to the fibronectin knock-out mouse (99; 100), reinforcing the concept that FAK is implicated in signaling induced by engagement of cells to ECM.

Upon integrin ligation and clustering, FAK is phosphorylated at Tyr-397 as well as at several additional sites within the kinase and the C-terminal domains (101-103) (Fig. 6). Tyr-397 is a major site of FAK auto-phosphorylation both in vivo and in vitro (104), and phosphorylation at Tyr-397 correlates with increased catalytic activity of FAK (101; 105). Therefore, phosphorylation of FAK at Tyr-397 is considered as a hallmark of integrin signaling.

Fig. 6: Linear structure of FAK showing its major domains, motifs and sites of tyrosine phosphorylation. The 4.1/JEF domain is also called FERM domain, and is thought to mediate interactions with proteins associated with the plasma membrane. The two proline-rich motifs mediate interactions with SH3-domain-containing proteins. Reproduced and adapted from reference (95).

1.2.2. FAK and downstream signaling

FAK interacts with a number of cytoskeletal and signaling proteins, including Src, PI3K, Grb2, p130^Cas and paxillin (95; 106), (Fig. 7). The finding that FAK is phosphorylated when cells are plated on fibronectin led to the hypothesis that FAK might transmit a signal within the cell through association with, and phosphorylation of, proteins that contain Src homology 2 (SH2) domains (90). The SH2 domain is a conserved region of about 100 amino acids, which specifically and selectively binds phosphotyrosyl proteins and promotes intra- and intermolecular protein-protein interactions (107). Indeed, it was subsequently demonstrated that phosphorylation of residue Tyr-397 enables the recruitment of several signaling proteins harboring SH2 domains. One such protein is the p85 kDa subunit of PI3K (108-110), which recruitment and activation leads to the activation of the kinase Akt. Another example is Src kinase (104). Src kinase then mediates phosphorylation on other sites on FAK, creating additional SH2-binding sites (101; 102). One such site is the phosphorylated residue Tyr-925 that can bind the SH2-domain of Grb2, leading to the activation of the MAP kinase ERK cascade (102; 111). Activation of these pathways involving the signaling proteins PI3K-Akt and ERK by engaged integrins is one of the main themas of this thesis project, and this topic is described more in detail below.

Tensin

Integrins ECM

Actin

1.2.3. Functions of FAK in the control of spreading and survival of cells

It is now acknowledged that integrin-induced FAK activation is involved in the regulation of cell proliferation (112-114), cell spreading/motility and cell survival (92; 93; 95).

Fibroblasts derived from FAK knock-out mice display deficient integrin-triggered spreading and migration (100). By contrast, overexpression of FAK either in CHO cells (115) or in FAK-deficient fibroblasts (116; 117) leads to increased spreading and/or migration of cells on fibronectin (115-117). Interestingly, overexpression of a FAK mutant lacking the Tyr-397 autophosphorylation site fails to rescue the effects of the inhibition or absence of FAK on cell spreading and/or migration (115-118). This suggests that auto-phosphorylation of FAK on residue Tyr-397 plays a crucial role in ECM-induced cell migration. Since, numerous studies have implicated FAK as a positive regulator of cell spreading and migration.

One of the main functions of FAK is the protection of cells against apoptosis. It was first reported that tyrosine phosphatase inhibitors block apoptosis subsequent to detachment from ECM (53). Subsequently, Frisch et al. (119) showed that constitutively active FAK mutants rescue MDCK cells from anoikis. Conversely, attenuation of FAK expression by treatment with antisense oligonucleotides induces apoptosis of tumour cell lines (120) and microinjection of peptides blocking binding of FAK to endogenous integrins or of anti-FAK antibody induces apoptosis of fibroblasts (121). It has also been reported that FAK-deficient cells are more susceptible towards serum withdrawal-induced apoptosis (122), and different dominant-negative FAK mutants have been shown to induce apoptosis in absence (122) or in presence of serum (123; 124). Now, it is generally accepted that FAK plays a critical role in suppressing anoikis and apoptosis caused by diverse inducers. Interestingly, it has been shown by several groups that FAK mutated on residue Tyr-397 is unable to promote survival of cells (119; 125; 126), suggesting that phosphorylation of Tyr-397 plays a critical role also in the pro-survival function of FAK. However, it has also been reported that the survival signal from the α5β1 integrin (in CHO cells overexpressing integrin α5 subunit) does not correlate with increased FAK phosphorylation (71), suggesting that pro-survival signaling from integrins may in some contexts occur independently of FAK.

In summary, FAK and phosphorylation of its tyrosine residue Tyr-397 have been shown to play central roles in integrin signaling leading to regulation of cell migration and survival.

Several proteins have been involved in this signaling, including PI3K, Akt and ERK.