Sistema umbral

In light of its localisation to cel 1-substratum contact points and activation by integrin engagement, FAK was predicted to have a primary role in cell motility and migration. This question was addressed directly by targetted FAK-gene deletion in mouse embryonic stem cells and generation of FAK-deficient mice (Furuta et a l, 1995). FAK deletion was embryonic lethal (E8.0-E8.5) and associated with severe morphogenetic defects in axial mesodermal tissues and the cardiovascular system. Several possibilities exist to explain the mesodermal defects, including slower migration o f FAK-null mesodermal cells. Additionally, reduced cell cycle length o f cells entering the primitive streak in FAK-null embryos or reduced survival o f mesodermal cells might plausibly contribute to the defects seen. It is o f interest that the overall phenotype o f FAK-null embryos resembles strongly that of fibronectin-deficient mice (George et a l, 1993). This data gives support to the concept of FAK playing a key role in integrin-

Stimulated signalling. Further support for this notion comes from results showing that homozygous deletion o f the murine p i integrin subunit gene or the a 5 integrin subunit gene, o f the specific integrin fibronectin receptor a 5 p l, result in similar early embryonic-lethal phenotypes (Stephens et al, 1995; Yang et al, 1993).

FAK-null fibroblasts have been propagated from E8.0 embryos and these cells exhibit migration but not proliferative defects in cell culture (Hi et a l, 1995b). PYK2 expression was noted to be increased in these cells; however, transient PYK2 overexpression did not reverse the migration defects o f these cells (Sieg et al, 1998). Somewhat unexpectedly, FAK-null cells had increased numbers o f focal adhesions suggesting that FAK might regulate focal adhesion turnover rather than assembly. Interestingly these cells were also noted to have normal phosphorylation levels o f focal adhesion-related proteins such as p i 30^^^ (Vuori et al, 1996), paxillin and cortactin (Hi et al, 1995a). This data suggests that other proteins are compensating for FAK in phosphorylating FAK-associated components, or that their phosphorylation is maintained via FAK-independent mechanisms.

Currently a good deal o f evidence (in addition to FAK-knockout studies) has accumulated to implicate a role for FAK in cell migration. Stable FAK overexpression in Chinese hamster ovary (CHO) cells was shown to cause enhanced cell migration (Cary et al, 1996, 1998). Reports o f overexpression o f FRNK leading to defective cell spreading and motility (Gilmore and Romer, 1996; Richardson and Parsons, 1996), and studies correlating elevated FAK expression with increased invasive potential o f human tumours (Owens et al, 1995), also point towards an important role for FAK in migration. In addition, enhanced FAK dephosphorylation achieved through overexpression o f a protein tyrosine phosphatase, was shown to antagonise FAK- mediated migration events (Tamura et al, 1999a, 1999b). Finally, it was recently shown that stable re-expression o f FAK in FAK-null cells was able to rescue both the morphological and integrin stimulated migration defects o f those cells (Sieg et al,

1.7.5 Role of FAK in cell proliferation.

FAK has already been linked with an extensive network o f signalling proteins that interface with growth regulation (reviewed in Clark and Brugge, 1995). These

include Src (Schaller et ai, 1994; Xing et al, 1994), the p85 subunit o f PI-3 kinase

(Chen and Guan. 1994; Guinebault et ai, 1995), the adaptor protein Grb-2 (Schlaepfer

et ai, 1994), and Csk (Sabe et al, 1994). These studies provided preliminary evidence suggesting a role for FAK in the regulation o f cell proliferation. Interestingly studies of FAK-null mice had shown no evidence o f effects on cell proliferation although it was remarked that FAK function may have been performed by other proteins in these cells

(Hi et al, 1995a). Subsequently several studies have been reported which give more

support to the notion that FAK is involved in cell cycle regulation. M icroinjection o f a glutathione-S-transferase fusion protein containing the COOH-terminal domain o f FAK (GST-Cterm). was shown to decrease DNA synthesis in HUVECs as compared to control-injected or non-injected cells (Gilmore and Romer, 1996). Overexpression of wild-type FAK was reported to accelerate G1 to S phase transition in NIH3T3 cells, whilst conversely overex press ion o f a dominant-negative FAK mutant AC 14 - which

competes with endogenous FAK - inhibited cell cycle progression at G1 phase (Zhao et

al , 1998). Recently, utilising a dominant-negative strategy again, it was established that

the activation o f .lun NH2-terminal kinase (.INK) by integrins is mediated by FAK and is

necessary for cell cycle progression (Oktay et al, 1999). The proposed mechanism by

which integrin^engagel%tit with ECM activated JNK was shown to depend upon the association o f FAK with Src and p l3 0 ^ ‘’^ and, subsequently, the recruitment o f Crk.

1.7.6 FAK and the regulation of apoptosis.

A number o f recent findings indicate a novel role for FAK in transducing

survival signals in anchorage dependent cells (Frisch et al, 1996; Hungerford et al,

1996; Crouch et al., 1996; Xu et al, 1996; Xiong et al, 1997; Tallett et al, 1996).

Expression o f a constitutively activated FAK construct in M DCK cells or the kératinocyte HaCat cell line conferred resistance to anoikis, a subset o f apoptosis arising

Additionally, in chicken embryo fibroblasts, apoptosis was induced by microinjection o f either an antibody to the FAT domain or a peptide corresponding to the FAK-binding site o f the (31-integrin cytoplasmic domain (Hungerford et al, 1996). Furthermore it was reported that attenuation o f FAK expression using anti sense oligonucleotides led to apoptosis in tumour cells (Xu, L.H. et al, 1998).

Previously it was reported that proteolysis may be an important mechanism o f the regulation o f FAK activity (Cooray et al, 1996). FAK proteolysis in thrombin- and collagen-activated platelets was shown to be mediated in part by the apoptotic protease calpain (Cooray et al, 1996). More recently these observations were extended in human cell lines undergoing apoptosis, and the caspase family o f apoptotic proteases were implicated directly in FAK cleavage (Wen et a l , 1997; Gervais et a l , 1998). Additionally, a comparison o f caspase-mediated proteolysis o f thirty-three signalling molecules during Jurkat cell apoptosis induced by Fas-L or etoposide, showed that FAK and two other molecules underwent cleavage with the same time course as for caspase activation (Widmann et al, 1998). Furthermore, focal adhesion disassembly and caspase-mediated cleavage of FAK was reported during apoptosis o f HUVECs induced by serum-deprivation (Levkau et al., 1998). An insight into how proteolytic cleavage o f FAK might be permissive for apoptotic signalling came from the studies o f Gervais et al, (1998). This group overexpressed chicken FAK in human cell lines and observed that FAK cleavage during apoptosis separates the tyrosine kinase domain from the FAT domain to create a FRNK-like polypeptide. These workers found that the carboxyl- terminal fragments which were generated were sufficient to suppress phosphorylation o f endogenous FAK in human cell lines. In view o f this, they proposed that the cleavage o f FAK by caspases might play an important role in the execution o f the suicide program by disabling the anti-apoptotic function o f FAK (Gervais et a l, 1998).

At present the mechanism by which FAK might participate in anti-apoptotic signalling is unclear, as is the apoptotic pathway triggered when integrin-FAK signals are interrupted. A recent study of apoptosis in anchorage-dependent cells has provided some important insights into FAK-mediated survival signalling (Hi et al, 1998). In this study multiple strategies were used to inactivate FAK and the p53 tumour suppressor protein in immortalised embryo-derived endothelial cells and fibroblasts.

The results showed that FAK transmitted survival signals from the ECM in both cell types and that in the absence o f FAK, a p53-regulated apoptotic pathway was activated, p53-dependent apoptosis was suppressible by mutant p53 and the small pro-domain caspase inhibitor ZVAD-fmk, but not by the large pro-domain caspase inhibitor CrmA (Hi g /a /.,1 9 9 8 ).

In summary, the evidence to date suggests that FAK is an early, selective and important target for small pro-domain caspases (e.g. caspase-3), and may play a key role in mediating survival signalling in anchorage-dependent cells.