[Frontiers in Bioscience 4, d102-113, January 15, 1999]

Current Issue

Accepted: 12/15/98

Send correspondence to:

Dr Jun-Lin Guan,
Department of Molecular Medicine,
Cornell University,
Ithaca, NY 14853, USA

E-mail: jg19@cornell.edu


Extracellular matrix (ECM), Focal adhesion kinase (FAK), Integrin, Signal transduction


Copyright © Frontiers in Bioscience, 1995


Leslie A. Cary and Jun-Lin Guan

Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA


1. Abstract
2. Introduction
3. Discussion
3.1. FAK-associated proteins and proposed functions
3.1.1. Src family members
3.1.2. PI 3-kinase
3.1.3. Grb2
3.1.4. p130Cas
3.1.5. Paxillin
3.2. Integrin-regulated functions mediated by FAK
3.2.1. Cell adhesion and spreading
3.2.2. Cell proliferation
3.2.3. Cell apoptosis
3.2.4. Cell migration
4. Acknowledgments
5. References


Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. These signaling events regulate such cellular processes as proliferation, apoptosis, migration and spreading. Focal adhesion kinase (FAK) is an important protein tyrosine kinase which mediates several integrin signaling pathways. Putative mechanisms of integrin-mediated FAK activation and localization to focal adhesions are discussed here. FAK interacts with a number of signaling and cytoskeletal proteins, including Src, phosphatidylinositol 3-kinase, Grb2, p130Cas and paxillin. Both the mechanisms and outcomes of these interactions are also presented. Finally, FAK’s roles in the regulation of several integrin-mediated cellular events are discussed, including the promotion of cell migration, proliferation and spreading, and the prevention of cell apoptosis.


Integrins are a family of cell surface receptors for extracellular matrix (ECM) proteins, such as fibronectin, vitronectin, collagen and laminin (1). They are heterodimeric glycoproteins, with at least 16 alpha subunits and 8 beta subunits identified so far which can combine to form at least 20 different receptors. In addition to serving as ECM adhesion molecules, integrins can also transduce biochemical signals into the cell to affect various proteins and second messengers, such as tyrosine kinases, serine/threonine kinases, lipid mediators, low molecular weight GTPases, and intracellular calcium fluxes. These signaling pathways regulate a variety of cellular functions, including spreading, proliferation, apoptosis and migration (2, 3, 4, 5). Defining and characterizing these signaling pathways is central to understanding the mechanisms of integrin-regulated cellular functions.

FAK (6, 7) is a critical mediator of integrin signaling. It is a cytosolic protein tyrosine kinase (PTK) which, in cells plated on an appropriate ECM substrate, is colocalized with integrins to focal adhesions. FAK is expressed in a variety of species, including human (8), rodent (7), chicken (6) and Xenopus (9, 10), indicating that it is evolutionarily conserved. In developing rodent embryos it is expressed beginning at approximately day 7.5 (11), and in adult tissues it is primarily detected in brain, lung, heart and testes (7, 11). Unlike many other cytosolic PTKs, FAK does not contain an SH2 (src-homology 2) or SH3 (src-homology 3) domain, but it does have SH2 and SH3 domain-interacting phosphotyrosines and proline-rich regions, respectively (see figure 1). FAK demonstrates increased kinase activity (12, 13) and tyrosine phosphorylation (7, 12, 14, 15, 16, 17) upon integrin activation in a variety of cell types, with the major site of phosphorylation identified as Y397 both in vivo and in vitro (18, 19, 20). Furthermore, phosphorylation at this site is mediated by FAK itself in an autophosphorylation mechanism. It is believed that FAK is the first identified member of a novel family of PTKs, based on the identification of a structurally related gene known as Pyk2, CAKbeta or RAFTK (21, 22, 23). In addition, other products of the FAK gene have been identified which are likely generated by alternative splicing, including a FAK-related non-kinase (FRNK), consisting of the C-terminal domain of FAK alone (24), and a series of FAK gene products which are preferentially expressed in brain tissues (25, 26, 27). Although its predominant role is in integrin signal transduction, FAK also responds to other extracellular stimuli (28); however, space does not permit a discussion of these pathways here.

Figure 1. FAK-interacting proteins. FAK contains a central protein tyrosine kinase domain which is flanked by regions showing no homology to previously identified proteins. Its major autophosphorylation site is Y397, which serves as a binding site for the SH2 domains of Src and PI 3-kinase (PI3K). The SH3 domain of Src also participates in its interaction with FAK by binding a proline-rich region upstream of Y397. Phosphorylation of Y925 by Src mediates FAK interaction with Grb2. A proline-rich region at amino acids 712-715 serves as the primary binding site for the SH3 domain of p130Cas, with a second proline-rich region at amino acids 878-881 contributing to this interaction. This second proline-rich region has been mapped as a binding site for the SH3 domain of Graf. The paxillin binding sequence in FAK overlaps completely with the focal adhesion targeting (FAT) sequence, and within this region lies the talin-binding sequence. Finally, a FAK sequence in its amino-terminal domain binds integrin beta subunits in vitro.

Although much is known about the role of FAK in integrin signaling, several crucial questions remain unanswered. First, the mechanism of FAK activation by integrins is not clear, although it is known to be specifically dependent on the integrin beta subunit (14, 29). There are currently two models describing beta integrin-mediated FAK activation. Direct binding of FAK to beta integrin cytoplasmic tails has been demonstrated in an in vitro system (30). However, concerns are raised based first on the lack of evidence for this interaction in other systems, and secondly on the differential mapping of the FAK binding site from the FAK activation site on the beta integrin (14). These results suggest that direct binding of FAK to the beta integrin is insufficient for its activation. A second model has been proposed involving the indirect association of FAK with the beta integrin through talin (31, 32). In vivo FAK association with talin as well as direct binding in vitro has been demonstrated (33). This model of FAK activation is believed to be the most likely based on several observations: first, FAK activation is dependent on the integrity of the actin cytoskeleton, as demonstrated by its disruption using cytochalasin D (16, 34); second, FAK binding to talin is not dependent on its catalytic activity; and finally, the talin binding site on the beta integrin overlaps with the site required for FAK activation (33).

Additionally, the mechanism of FAK localization to focal adhesions is not entirely clear. The FAK sequence required for this event was defined as the focal adhesion targeting (FAT) sequence, a 159 amino acid stretch located in the C-terminal domain of the protein (35) which was later redefined to a 148 amino acid stretch (36). Significant data exists to suggest that FAK localization is mediated by binding to paxillin through this FAT sequence. At least 17 different truncation mutants and 6 different point mutants of FAK have correlative abilities to localize to focal adhesions and bind paxillin (36, 37). The only data which sheds doubt on this model is that a FAK construct with a C-terminal epitope tag is localized to focal adhesions but does not bind paxillin (36). It is possible that this interaction is only destabilized by this epitope tag; however, because of this result it remains unclear whether paxillin mediates FAK localization to focal adhesions.


3.1. FAK-associated proteins and proposed functions

The major site of FAK phosphorylation, Y397, serves as a binding site for the SH2 domains of Src (19, 20) and phosphatidylinositol 3-kinase (PI 3-kinase) (38). This phosphotyrosine is required for FAK functions in many different integrin signaling pathways, indicating the importance of FAK association with these two proteins. FAK associates with a number of other signaling and cytoskeletal molecules (see figure 1) which will be described here, including Grb2, p130Cas(crk-associated substrate), and paxillin. Both the mechanisms of FAK association with these proteins and the functional outcomes of these interactions in integrin-initiated signaling pathways will be discussed. Tensin is also believed to associate with FAK based on its tyrosine phosphorylation correlative with that of FAK (39, 40) which is likely mediated by Src (41). Tensin demonstrates properties of both an actin cytoskeletal protein and an integrin-activated signaling molecule (39, 40, 42, 43). However, neither the mechanism nor the functional outcome of FAK’s putative interaction with tensin is clear. FAK also binds a recently identified protein called Graf (GTPase regulator associated with FAK), which is mediated by the SH3 domain of Graf and a proline-rich region on FAK (44). The demonstrations that Graf can localize with the actin cytoskeleton and that its GAP domain stimulates the GTPase activity of Rho and Cdc42 suggest that Graf may regulate the actin cytoskeleton through these proteins (44). Furthermore, Graf is believed to be a substrate of mitogen-activated protein (MAP) kinase, suggesting that it may respond to mitogenic or other stimuli (45). However, the functional role of FAK/Graf association remains a mystery, particularly since Graf is not expressed in most of the cell lines where FAK’s role has been well characterized (45). Therefore the putative roles of both tensin and Graf in FAK signaling pathways will not be addressed here.

3.1.1. Src family members

The Src family of cytosolic protein tyrosine kinases (46) function intimately with FAK in integrin signaling pathways. Furthermore, the original identification of FAK as a protein with increased tyrosine phosphorylation in v-Src-transformed cells (6, 13, 47) suggests that FAK may mediate some of the oncogenic functions of Src. The major autophosphorylation site on FAK, Y397, serves as a binding site for the Src SH2 domain (19, 20), and in fact association of FAK with Src family members has been demonstrated in a number of in vivo and in vitro systems (19, 20, 48, 49, 50). Although Y397 mediates the principle interaction of FAK with Src, a proline-rich region upstream of this phosphotyrosine may contribute to their association by binding the SH3 domain of Src (51). Consistent with this observation, the binding of a Src-SH2 construct to FAK is weaker than that of a Src-SH3-SH2 construct (49).

The outcome of Src binding to FAK is the phosphorylation of several tyrosine residues in FAK as well as in several FAK-associated proteins. In addition to Y397, FAK tyrosines 407, 576, 577, 861 and 925 have been mapped as phosphorylation sites. Of these, Y397 is the only residue clearly phosphorylated by FAK itself, while the others are phosphorylated by Src. The phosphorylation of Y576 and Y577 in the kinase domain of FAK positively regulates FAK catalytic activity (52). The roles of Y407 phosphorylation (52) or Y861 phosphorylation (53) are not clear, but are suggested to mediate binding to SH2 domains based on their neighboring residues. Finally, phosphorylated Y925 has been mapped as a binding site for the SH2 domain of the Grb2 adaptor protein which is believed to partially mediate activation of the Erk (extracellular-regulated kinase) family of MAP kinases (see below).

Of the known FAK-associated proteins, paxillin and p130Cas tyrosine phosphorylation are mediated at least in part by FAK-associated Src. Their phosphorylation in response to FAK expression is dependent on FAK Y397 (54, 55, 56), and while it is increased in Csk-/- cultured cells and reduced in Src-/- cells, it is not affected in FAK-/- cells (41, 55, 57). Because Pyk2 expression is increased in FAK-/- cells (58), it is possible that this homolog or other proteins may compensate for the absence of FAK in some functions. Although paxillin phosphorylation has been demonstrated by FAK in vitro (54, 59), because a recombinant source of FAK was not used it is not clear whether FAK-associated Src might be responsible for this phosphorylation. On the other hand, it is clear that p130Cas can be directly phosphorylated by FAK in vitro at the Src-binding YDYVHL sequence (60); therefore, the outcome of this event is the recruitment of Src to allow for complete p130Cas phosphorylation by this kinase. Src is therefore the kinase necessary for most if not all of FAK-promoted paxillin and p130Cas tyrosine phosphorylation in these complexes.

The outcome of Src phosphorylation of FAK or associated proteins is not always clear. FAK Y397 is crucial for many of the established biological roles of FAK, including cell migration (50), cell cycle progression (61) and prevention of anoikis, a form of detachment-induced apoptosis (62). More specifically (see below), Src binding to Y397 mediates phosphorylation of FAK Y925, binding to Grb2, and partial Erk activation by integrins. Substantial evidence demonstrates that Src binding to Y397 is necessary for FAK-promoted cell migration through its phosphorylation of p130Cas in this complex. Likewise, it is suggested that Src-mediated phosphorylation of paxillin in this complex mediates the rescue of FRNK-inhibited cell spreading. Characterization of Src’s role by binding FAK Y397 is further complicated by the binding of an additional protein (PI 3-kinase) to this site. Clearly, Src is crucial for many of FAK’s functions in integrin signal transduction through its phosphorylation of FAK and associated proteins.

3.1.2. PI 3-kinase

PI 3-kinase is an important lipid kinase which has recently been demonstrated to play a role integrin signal transduction. Some of its lipid products can act as second messengers to regulate protein kinase C (PKC) family members (63, 64) as well as the Akt kinase (65). PI 3-kinase is a heterodimeric enzyme, comprised of a 110 kDa catalytic subunit (p110) and an 85 kDa regulatory subunit (p85). In addition to its p110 binding site, the p85 subunit contains SH2 and SH3 domains which mediate its interactions with other cellular proteins. Association of FAK with PI 3-kinase in response to integrin activation has been demonstrated in both platelets (66) and fibroblasts (67). In addition, FAK/PI 3-kinase association in fibroblasts is stimulated by cell treatment with platelet-derived growth factor (PDGF) (68), suggesting a mechanism of cross-talk between integrin and growth factor signaling pathways. However, while it was suggested that p85 binding to FAK in platelets is dependent on the SH3 domain of p85, substantial evidence demonstrates that in fibroblasts it is mediated by FAK tyrosine phosphorylation. Furthermore, FAK Y397 was mapped as both an in vitro and in vivo binding site for PI 3-kinase through one or both SH2 domains of p85 (38). As discussed above, this phosphotyrosine also serves as a Src binding site; neither the mechanisms nor ramifications of two proteins binding to the same site on FAK are clear.

The requirement for FAK Y397 for its roles in cell proliferation, apoptosis and migration suggests that PI 3-kinase binding to FAK may be involved in any or all of these events. Because substantial data demonstrate a role for PI 3-kinase in preventing cell apoptosis through its downstream mediator Akt (69), the possibility that FAK/PI 3-kinase association regulates apoptosis should be emphasized, although there is no specific information available about this putative pathway. A role for PI 3-kinase in regulating cell motility has also been demonstrated in a number of studies. Growth factor-stimulated cell migration is dependent on PI 3-kinase (70, 71, 72, 73, 74), as is integrin alpha6beta4-stimulated carcinoma cell invasion (75) and chinese hamster ovary (CHO) cell migration on fibronectin (76). Cell migration stimulated by Rac or Cdc42 is also dependent on PI 3-kinase, and a constitutively active PI 3-kinase construct promotes cell migration on collagen (77). In addition, treatment of NIH 3T3 cells with purified PI(3,4,5)P3, a product of PI 3-kinase, increases their migration (78). Finally, recent evidence demonstrates specifically that PI 3-kinase association with FAK plays a positive role in cell migration. A FAK point mutation proximal to Y397 which selectively disrupts its binding to p85 but not Src abolishes FAK-promoted cell migration (76). The putative role of FAK/PI 3-kinase binding in other integrin-regulated signaling pathways will likely be an area of future study.

3.1.3. Grb2

The demonstrations that Grb2 binds directly to FAK at Y925 in response to integrin activation and that this site is phosphorylated by Src are intriguing (79, 80). They suggest that FAK acts in a manner analogous to a growth factor receptor in order to recruit Grb2 and Sos to the plasma membrane and initiate a Ras/MAP kinase pathway. However, while FAK expression in HEK 293 cells enhances fibronectin-stimulated Erk activation, this effect is dependent on FAK Y397 but only partially on FAK Y925 (81, 82). It is therefore believed that Grb2 binding to FAK mediates only a minor pathway downstream of FAK to activate Erks, with the major pathway initiated by FAK/Src-mediated tyrosine phosphorylation of Shc and its association with Grb2 (82) and another minor pathway mediated by p130Cas phosphorylation and recruitment of an SH2 domain-containing protein (83). In addition, a role for FAK/Grb2 binding in FAK-regulated cell migration has been ruled out (56), and although FAK/Grb2 association is detected in human monocytes treated with macrophage colony-stimulating factor (M-CSF), the role of their interaction in this system is not clear (84). Presently, therefore, the only established role of Grb2 binding to FAK is in the partial regulation of MAP kinases by integrins.

3.1.4. p130Cas

P130Cas was identified as a protein with increased tyrosine phosphorylation in cells transformed by either the v-src or v-crk oncogenes (47, 85, 86, 87), and as expected it also demonstrates association with both of these proteins (85, 88). Cloning of p130Cas cDNA demonstrated that it is an adaptor protein, containing one SH3 domain and multiple tyrosines which are in consensus for SH2-binding sites (85). Most importantly, p130Cas contains multiple tyrosines in the YxxP motif, which are consensus Crk SH2-binding sites, and this YxxP repeat region within p130Cas is referred to as the substrate domain. Binding to Src is mediated by two independent sites within the C-terminal domain of p130Cas: a Src SH2-binding phosphotyrosine (Y762) in the sequence YDYVHL which is outside of the substrate domain, and a Src SH3-binding proline-rich region (89). Two homologs of p130Cas have been identified, each by two independent groups. The first was named both Efs (90) and Sin (91), while the second was named both HEF-1 (92) and CasL (93). Both homologs are structurally similar to p130Cas, containing an SH3 domain as well as multiple YxxP motifs. Efs/Sin is believed to play a role in some aspect of signaling by Src family members, while HEF-1/CasL is suggested to play roles in T cell receptor-mediated functions and/or regulation of the actin cytoskeleton. The relationship between the functions of p130Cas and these homologs remains to be determined.

An important role has been demonstrated for p130Cas in integrin signal transduction pathways. It is localized to focal adhesions in rat fibroblasts (87, 94) as well as in COS cells plated on fibronectin, where complete p130Cas localization is dependent on its SH3 domain, substrate domain, and Src-binding sites (95). Integrin activation induces tyrosine phosphorylation of p130Cas on various ECM proteins (86, 87, 94, 96), as well as in cells activated by anti-integrin antibodies (96, 97, 98). Further evidence for its role in integrin signal transduction comes from the demonstration that p130Cas binds FAK directly through its SH3 domain (88, 94, 99). There are two proline-rich regions in FAK which participate in binding to p130Cas. The major p130Cas-binding site, identified first, is located at amino acids 712-715 (99). A second site at amino acids 878-881 also contributes to this interaction, and both sites must be mutated in order to completely abolish p130Cas binding to FAK (94, 100). Interestingly, p130Cas is a substrate for several protein tyrosine phosphatases, including PTP-PEST (101), the Yersinia YopH protein (102) and PTP1B (103, 104). At least in the case of PTP1B, the SH3 domain of p130Cas mediates direct binding to the phosphatase, which may indicate a competitive interaction between FAK and PTP1B for binding p130Cas.

Because p130Cas phosphorylation is increased by integrin activation and other FAK-activating stimuli and because it binds directly to FAK, a reasonable speculation is that FAK mediates p130Cas tyrosine phosphorylation. Indeed, FAK overexpression in either rat fibroblasts (55) or CHO cells (56) increases p130Cas phosphotyrosine levels. As discussed above, Src is primarily responsible for tyrosine phosphorylation of p130Cas in this complex (55). Although FAK does phosphorylate p130Cas directly, this event allows for Src binding to p130Cas and subsequently its complete phosphorylation of p130Cas at sites in the substrate domain (60). This model predicts that integrin-mediated phosphorylation of p130Cas by the FAK/Src complex results in its association with Crk. In fact, in vivo p130Cas/Crk association as well as in vitro binding of p130Cas to several signaling proteins has been demonstrated by cell adhesion (55). In addition, several protein tyrosine phosphatases interact with p130Cas, as discussed above, and are believed to be responsible for the downregulation of these p130Cas-mediated signaling pathways through their dephosphorylation of p130Cas and/or associated proteins.

The functional role of p130Cas binding to FAK is in the regulation of cell motility, as demonstrated in several ways in CHO cells overexpressing FAK (56). Furthermore, the mechanism of p130Cas function in this system is believed to involve tyrosine phosphorylation of p130Cas by FAK-associated Src and subsequent recruitment of an SH2 domain-containing signaling molecule to p130Cas. Correlative with this, p130Cas has also been demonstrated to play a role in COS cell migration, as expression of either p130Cas or Crk increases cell migration, which is dependent on their respective binding sites (105). Whether FAK/Src/p130Cas-promoted migration in CHO cells is mediated by p130Cas/Crk association remains to be determined. Other SH2 domain-containing proteins bind p130Cas in a FAK- or adhesion-dependent manner, such as PI 3-kinase (55, 56), and therefore could also regulate cell motility by p130Cas.

If and when p130Cas does regulate cell migration through Crk, the mechanisms involved are not clear. Interestingly, either cell attachment or Src transformation increases the amount of FAK/p130Cas complexes in detergent-insoluble subcellular fractions (100). Similarly, Crk transformation increases the amount of cytoskeletal-associated p130Cas (106). These results suggest that p130Cas may control cell motility through regulation of the cytoskeleton. The Crk-binding protein DOCK180 is implicated in this mechanism, as it is suggested to regulate actin cytoskeleton dynamics (107, 108). Because the FAK/Src/p130Cas complex regulates the Erk family of MAP kinases (83), and Erks have been demonstrated to directly phosphorylate and activate myosin light chain kinase (109), regulation of the actin cytoskeleton in this way is another possibility. However, a role for Erks in FAK-promoted CHO cell migration has been ruled out, because FAK overexpression does not promote Erk activation, nor does a specific MEK inhibitor affect cell migration in this system (56). Furthermore, COS cell migration by p130Cas expression is not inhibited by coexpression of dominant-negative Ras (105). Erks are therefore not believed to mediate cell migration by p130Cas. Another possible mechanism of p130Cas-mediated cell migration is through the Rho family of GTPases, since Crk is suggested to mediate some of its cell morphological effects through activation of Rho signaling pathways (110) which are known to regulate specific actin cytoskeletal functions (111, 112). It should be noted that the major pathway downstream of Crk that has been identified results in activation of the JNK (Jun N-terminal kinase) family of MAP kinases (113, 114); however, there is little evidence to suggest that JNK may regulate cell motility. In conclusion, p130Cas clearly regulates cell motility through its associations with FAK and Crk; however, while many pathways downstream of these proteins have been identified, it is not clear which are mediating the effects on cell motility.

3.1.5. Paxillin

Paxillin was first identified as a cytoskeletal protein with increased tyrosine phosphorylation in v-Src transformed fibroblasts (115). Its roles in integrin signal transduction and/or cytoskeletal organization are demonstrated based on its localization to focal adhesions, binding to vinculin, and tyrosine phosphorylation concomitantly with FAK upon integrin activation (116, 117). The latter result also suggests that paxillin may be a substrate of FAK or a FAK-associated kinase. Analysis of paxillin cDNA (117) reveals many putative binding sites for other proteins, including several SH2 domain-binding phosphotyrosines, SH3 domain-binding proline-rich regions, and four LIM domains which have been shown to mediate protein dimerization (118, 119). In various in vitro or in vivo binding assays, paxillin binds vinculin (116, 117), integrin beta subunits (30, 120), Src family members (54, 121), FAK (36, 37, 117), Csk (54), Crk (54, 122) and the tyrosine phosphatase PTP-PEST (123). Because of its lack of enzymatic activity, paxillin is generally believed to act as a scaffolding protein in focal adhesions by mediating interactions with other signaling and/or cytoskeletal proteins.

The mechanism of paxillin localization to focal adhesions is not clear, although at least two binding proteins have been ruled out (124). In this study, it was demonstrated that the third LIM domain of paxillin (LIM3) was necessary for paxillin localization, with LIM2 possibly contributing to this effect. However, both the vinculin and FAK binding sites were mapped to paxillin’s LD motifs (short stretches named for their repeats of leucine and aspartic acid), sequences which are well separated from LIM2 and LIM3. Therefore, paxillin is targeted to focal adhesions by a LIM domain-mediated interaction and not by binding vinculin or FAK, which is consistent with the model of FAK localization to focal adhesions by its binding to paxillin. The protein(s) which bind to these LIM domains to mediate paxillin localization have not yet been identified. Furthermore, phosphorylation of various serine and threonine residues within LIM2 and LIM3 affects paxillin localization in a transient manner (125), but the kinase(s) responsible for these phosphorylation events are also not known.

The roles of FAK association with paxillin are not completely clear. Substantial evidence demonstrates that paxillin recruits FAK to focal adhesions by its direct binding to the FAT sequence, thus in this model, paxillin is upstream of FAK. However, many studies suggest that paxillin may also be a downstream mediator of FAK signal transduction pathways. Because tyrosine phosphorylation of paxillin occurs concomitantly with FAK, it is believed to occur in a FAK-dependent manner. Indeed, increased tyrosine phosphorylation of paxillin has been demonstrated as a result of FAK overexpression in CEF cells (54). However, several lines of evidence suggest that Src rather than FAK is responsible for paxillin phosphorylation (41, 54, 57). In vitro phosphorylation of paxillin by FAK has been demonstrated in two independent studies, with a major site of phosphorylation mapped to Y118 in both (54, 59). However, in neither study was a recombinant source of FAK used; thus it is possible that phosphorylation of paxillin Y118 is mediated by FAK-associated Src. In any case, phosphorylation of paxillin at Y118, whether by FAK or by Src, is not necessary for efficient binding to FAK (59), but is suggested to create a binding site for Crk, as this tyrosine fits the consensus YxxP motif (YSFP in paxillin) for binding the Crk SH2 domain. Indeed, paxillin phosphorylated as a result of FAK overexpression does demonstrate increased in vitro binding to a Crk SH2 construct (54), and stable in vivo association of paxillin and Crk has also been demonstrated by coimmunoprecipitation in Crk-transformed cells (122). The biological role of paxillin Y118 phosphorylation and binding to Crk is not clear, although roles in both paxillin localization to focal adhesions (59, 125) and cell adhesion to fibronectin (125) have been ruled out by studies with a paxillin Y118F mutant. Recently, a direct interaction between paxillin and PTP-PEST has been demonstrated (123); dephosphorylation of paxillin and/or associated proteins by this phosphatase is suggested as an important regulatory mechanism of paxillin-mediated signaling pathways.

Identification of a functional role for paxillin downstream of FAK is complicated by the lack of identification of a functional role for paxillin itself. Recently, expression of paxillin mutants targeting the serine/threonine phosphorylation sites in the LIM domains were shown to affect cell adhesion to fibronectin (125). To date, this is the best established functional role for paxillin, although it is not clear whether its association with FAK and/or Src may be involved in this process. Because paxillin phosphorylation is believed to be mediated by Src, and because FAK’s Src-binding site (Y397) is crucial for many of its established functional roles, including cell proliferation, migration, and prevention of apoptosis, it is possible that paxillin mediates some of these events by the FAK/Src complex. However, paxillin tyrosine phosphorylation by FAK overexpression in CHO cells is only partially dependent on Y397 (50). Furthermore, the role of FAK Y397 in this system is believed to be necessary for Src-mediated phosphorylation of p130Cas (56) and for an independent PI 3-kinase mediated pathway (76). Therefore we conclude that paxillin at best is only partially responsible for FAK-promoted cell migration. More convincing is the suggestion that FAK/Src-mediated phosphorylation of paxillin mediates the rescue of FRNK-inhibited cell spreading, since paxillin phosphorylation correlates completely with this phenotype (126). Paxillin is therefore believed to localize FAK to focal adhesions, and may play additional roles downstream of FAK in the regulation of cell spreading or other events.

3.2. Integrin-regulated functions mediated by FAK

Many FAK-associated proteins have been described, and in some cases the biochemical signal transduction pathways resulting from these interactions have been identified. Furthermore, many functional roles for FAK in integrin signaling processes are known. It was originally predicted that FAK plays a role in integrin-mediated cell adhesion, spreading or focal adhesion formation; however, little evidence exists to date to support this hypothesis. Instead, roles for FAK have been identified in the positive regulation of cell proliferation and cell migration, and the prevention of cell apoptosis. The signaling pathways responsible for these events will be described here (see figure 2).

Figure 2. Integrin signal transduction pathways mediated by FAK. FAK has been demonstrated to play a role in several different integrin-mediated cellular events, including the promotion of cell migration, proliferation and spreading, and the prevention of cell apoptosis. Both established and putative signaling pathways downstream of FAK which regulate these processes are shown here. Question marks (?) indicate unclear mechanisms and/or pathways. Two pathways have been described downstream of FAK which promote cell migration: the first occurs through Src-mediated phosphorylation of FAK-associated p130Cas and may require Crk downstream of p130Cas, and the second is mediated by PI 3-kinase (PI3K) association with FAK. Three independent pathways downstream of the FAK/Src complex regulate the Erk family of MAP kinases, which are predicted to regulate cell proliferation. The major pathway involves the promotion of Grb2/Shc association by an unknown mechanism, with minor pathways occurring through FAK binding to Grb2 and p130Cas. FAK clearly prevents apoptosis, although the mechanisms by which it does so are less clear than the pathways described above. It is suggested that PI3K and possibly Akt are downstream of FAK in this pathway. A separate pathway involving PLA2, PKC and p53 has also been described. Finally, FAK-promoted cell spreading is believed to occur through Src-dependent phosphorylation of paxillin in this complex.

3.2.1. Cell adhesion and spreading

FAK was identified as a protein tyrosine kinase which demonstrates increased kinase activity and tyrosine phosphorylation in response to integrin activation, e.g. by cell attachment to ECM proteins. Because of the strong correlation between FAK regulation and these integrin-dependent events, it was predicted that FAK mediates these functions. However, there is little evidence to support this hypothesis, although many studies have examined this possibility. There is no direct evidence to demonstrate a role for FAK in regulating integrin-mediated cell adhesion. Cultured cells from FAK-/- cells show little or no differences in adhesion to fibronectin compared to FAK+/+ cells (57); likewise, CHO cells which stably overexpress FAK demonstrate no altered cell adhesion to fibronectin (50). However, some studies do indicate that FAK may mediate cell spreading on ECM proteins. Although FAK expression in various cultured cells does not affect cell morphology or spreading (35, 50), FAK-/- cells are more poorly spread when plated on fibronectin than their FAK+/+ counterparts (57). Less direct evidence for a FAK role in cell spreading comes from studies with the C-terminal FAK-related non-kinase, FRNK (24). Expression of FRNK in chicken embryo fibroblast cells results in delayed cell spreading on fibronectin, i.e. FRNK-expressing cells spread more slowly, but eventually their morphology is indistinguishable from that of control cells (127). Furthermore, this reduced cell spreading correlates to some degree with reduced tyrosine phosphorylation of FAK and can be overcome by coexpression of wild-type FAK. From these results it is believed that FRNK acts as a competitive inhibitor of FAK, and therefore this phenotype reflects a FAK-mediated function. In addition, cell spreading in this system was shown to correlate with paxillin but not tensin phosphorylation (126). It is therefore suggested that phosphorylation of paxillin or other proteins by the FAK/Src complex regulates cell spreading.

3.2.2. Cell proliferation

Several studies demonstrate a role for FAK in regulating cell growth. This was first suggested based on studies using a FRNK-related FAK construct. When microinjected into cells this construct inhibits DNA synthesis, presumably through competitive inhibition of FAK (128). Recently, inducible expression of FAK was demonstrated to positively regulate cell cycle progression (61). While wild-type FAK increases DNA synthesis and accelerates the G1/S transition, a dominant-negative FAK construct inhibits these steps, and these effects correlate with expression of cyclin D1 and the cdk inhibitor p21. Furthermore, these FAK-mediated cell cycle effects are dependent on Y397 and are believed to require Src and/or PI 3-kinase binding to this site. In addition, FAK is predicted to mediate integrin-dependent regulation of the Erk family of MAP kinases through several pathways (81, 82, 83), and these pathways are suggested to act synergistically with mitogenic signaling pathways to regulate cell growth (129, 130, 131, 132). Interestingly, FAK-/- cells do not demonstrate reduced proliferation (57), which is likely a result of compensation by the FAK-related protein Pyk2 (58). Together, these results demonstrate that FAK positively regulates cell cycle progression and cell proliferation. Future studies will likely determine the mechanisms of FAK function in these pathways.

3.2.3. Cell apoptosis

In several studies FAK has been demonstrated to protect cells from a form of apoptosis known as anoikis, which is induced by cell detachment from the ECM. Expression of an activated, membrane-targeted FAK construct called CD2FAK (18) in epithelial cells prevents anoikis (62). Conversely, inhibition of FAK either by treatment of tumor cell lines with FAK antisense oligonucleotides (133) or by microinjection of CEF cells with an anti-FAK monoclonal antibody (134) induces apoptosis. In addition, various cell lines induced to undergo apoptosis demonstrate concomitant FAK proteolysis (135, 136, 137), and several caspases, apoptosis-specific proteolytic enzymes, have been shown to cleave FAK at specific sites in vitro (136, 138). Cleavage of FAK is predicted to generate FRNK-like polypeptides, so that not only are the levels of full-length FAK reduced in apoptotic cells, but they are replaced by a competitive inhibitor of FAK. The mechanisms of cell survival by FAK are not clear. Because Y397 of FAK is required for its prevention of anoikis, FAK association with Src and/or PI 3-kinase is predicted to be necessary (62). Recent evidence also suggests that FAK prevents apoptosis through a pathway involving phospholipase A2 (PLA2), PKC and p53, although the pathways from FAK to these proteins are not clear (139).

3.2.4. Cell migration

Perhaps the best defined role for FAK in integrin-mediated cellular functions is in the promotion of cell migration. This was first suggested by the demonstration that a tyrosine kinase was required for endothelial cell migration and that the activity of FAK was correlated with this event (140). More direct evidence for FAK’s role in cell motility comes from several different studies. Inhibition of FAK by microinjection of a FRNK-related construct results in decreased endothelial cell motility (128). A FAK homozygous knockout in mice is embryonic lethal, with embryos showing abnormalities in mesoderm development, and cells cultured from these FAK-/- embryos display decreased motility in vitro (57). Conversely, stable overexpression of FAK in CHO cells results in increased cell motility on fibronectin, which is dependent on both Y397 (50) and the p130Cas proline-rich binding site (56). FAK is believed to regulate CHO cell migration through at least two distinct pathways: one involving tyrosine phosphorylation of FAK-associated p130Cas by Src, which binds FAK at Y397 (56), and the other involving a distinct pathway initiated by PI 3-kinase binding to FAK Y397 (76). Interestingly, the FAK homolog Pyk2 can substitute for FAK in the regulation of MAP kinases but not in the regulation of cell migration (58).

The implications for FAK’s role in integrin-mediated cell motility are several. The first is in the regulation of cell migration steps required for proper embryonic development at approximately day 8 of mouse development. FAK expression is detected starting at about day 7.5 (11), while FAK-/- mice are embryonic lethal at about day 8.5 with defects that suggest abnormalities in mesoderm migration (57). Another is in tumor cell invasion and metastasis because FAK expression is correlated with more invasive human tumors (8, 141), although it is not clear whether FAK expression in these tumors is a cause or a consequence of their increased invasiveness. Finally, FAK expression correlates with keratinocyte migration in repairing burn wounds, suggesting that FAK-mediated motility may also be important for wound healing processes (142).


Research in the authors’ laboratory is supported by NIH grants R01GM48050 and R01GM52890. Jun-Lin Guan is an Established Investigator of the American Heart Association. We thank Dong Cho Han, Molly Ingersoll, Heinz Reiske and Ji-He Zhao for critical reading of the manuscript.


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