an important `molecular switch' for the induction of migration signals via its binding to the SH2/SH3- adapter protein Crk (Vuori et al., 1996). The expression of ...
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Oncogene (2000) 19, 5606 ± 5613 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc
Focal Adhesion Kinase: a regulator of focal adhesion dynamics and cell movement J Thomas Parsons*,1, Karen H Martin1, Jill K Slack1, Joan M Taylor1 and Scott A Weed1,2 1
Department of Microbiology, Health Science Center, University of Virginia, Charlottesville, Virginia, VA 22908, USA
Engagement of integrin receptors with extracellular ligands gives rise to the formation of complex multiprotein structures that link the ECM to the cytoplasmic actin cytoskeleton. These adhesive complexes are dynamic, often heterogeneous structures, varying in size and organization. In motile cells, sites of adhesion within ®lopodia and lamellipodia are relatively small and transient and are referred to as `focal complexes,' whereas adhesions underlying the body of the cell and localized to the ends of actin stress ®bers are referred to as `focal adhesions'. Signal transduction through focal complexes and focal adhesions has been implicated in the regulation of a number of key cellular processes, including growth factor induced mitogenic signals, cell survival and cell locomotion. The formation and remodeling of focal contacts is a dynamic process under the regulation of protein tyrosine kinases and small GTPases of the Rho family. In this review, we consider the role of the focal complex associated protein tyrosine kinase, Focal Adhesion Kinase (FAK), in the regulation of cell movement with the emphasis on how FAK regulates the ¯ow of signals from the ECM to the actin cytoskeleton. Oncogene (2000) 19, 5606 ± 5613. Keywords: integrins; protein tyrosine kinase; adhesion; migration; phosphorylation Introduction Adhesion of cells to the extracellular matrix (ECM) is mediated by the integrin family of heterodimeric receptors (Burridge and Chrzanowska-Wodnicka, 1996; Burridge et al., 1988). Engagement of integrin receptors with their extracellular ligands leads to the formation of well-de®ned structures linking the ECM and cytoplasmic actin cytoskeleton. In tissue culture cells, points of adhesion, termed focal adhesions, are visualized as sites of close membrane contact with the substrate and anchor a highly bundled and crosslinked actin stress ®ber network (Burridge et al., 1988; Craig and Johnson, 1996). It is now clear that sites of ECMintegrin adhesion are in fact, dynamic, often heterogeneous structures, varying in size and organization (Zamir et al., 1999, 2000). In motile cells, sites of adhesion within ®lopodia and lamellipodia are rela-
*Correspondence: JT Parsons, Department of Microbiology, PO Box 800734, Health Sciences Center, University of Virginia, Charlottesville, VA 22908, USA 2 Current address: Department of Basic Sciences and Oral Research, School of Dentistry, Box C286, University of Colorado Health Sciences Center, 4200 Ninth Ave., Denver, CO 80262, USA
tively small and transient and are referred to as `focal complexes'. In contrast, the large, stable sites of adhesion underlying the body of the cell and localized to the ends of actin stress ®bers are referred to as `focal adhesions' (Hall, 1998). Signal transduction through focal complexes and focal adhesions has been implicated in the regulation of a number of key cellular processes, including growth factor induced mitogenic signals, cell survival and cell locomotion (Burridge and Chrzanowska-Wodnicka, 1996; Schwartz et al., 1995). The dynamic regulation of cell adhesion is of particular importance when cells move in response to a stimulus, either an immobilized ligand, e.g., ECM proteins (haptotaxis) or a soluble ligand, e.g., growth factors or cytokines (chemotaxis) (Horwitz and Parsons, 1999; Lauenburger and Horwitz, 1996). Focal adhesions serve as sites for force transmission for the cell against the rigid substratum, force provided by contraction of the actin-myosin stress ®ber network (Burridge and Chrzanowska-Wodnicka, 1996; Horwitz and Parsons, 1999). Newly generated `focal complexes' formed at the leading edge of the cell provide new adhesive contacts and thus provide `directionality' for cell movement (Horwitz and Parsons, 1999). The coordinated regulation of the formation and turnover of adhesion complexes is central to how cells move in response to dierent signals. The formation and remodeling of focal contacts is a dynamic process under the regulation of protein tyrosine kinases and small GTPases of the Rho family (Burridge and Chrzanowska-Wodnicka, 1996; Horwitz and Parsons, 1999). In this review we consider the role of the focal complex associated protein tyrosine kinase, Focal Adhesion Kinase (FAK), in the regulation of cell movement with the emphasis on how FAK regulates the ¯ow of signals from the ECM to the actin cytoskeleton. Cell adhesion and migration ± the protein tyrosine kinase connection Cellular attachment is mediated by the direct ligation of integrin extracellular domains to de®ned sequences within ECM proteins (Burridge and ChrzanowskaWodnicka, 1996; Schwartz et al., 1995). The clustering and activation of integrin receptors induces the formation of cytoplasmic membrane proximal adhesions (Burridge et al., 1988; Calderwood et al., 2000; Schwartz et al., 1995). The organization of these adhesions is complex, but includes a number of abundant cytoskeletal proteins (vinculin, talin, alpha actinin) as well as several cytoplasmic protein tyrosine
FAK and cell migration JT Parsons et al
kinases, including members of the Src family and FAK (Burridge et al., 1988). Targeting of protein tyrosine kinases to focal adhesions leads to their activation and the subsequent tyrosine phosphorylation of multiple focal complex-associated proteins (see below). Inhibitor studies clearly indicate that tyrosine phosphorylation plays an important role in the overall organization of adhesion complexes and their dynamic regulation (Burridge et al., 1988; Craig and Johnson, 1996). FAK and FAK binding partners One of the most prominent alterations observed upon integrin clustering is the phosphorylation on tyrosine of a variety of proteins, including the protein tyrosine kinase, FAK (Guan et al., 1991; Kornberg et al., 1992; Lipfert et al., 1992; Schaller et al., 1992). FAK is the prototype for a family of non-receptor protein tyrosine kinases. FAK is expressed in most tissues and cell types and is evolutionarily conserved across species (Fox et al., 1999; Hanks et al., 1992; Palmer et al., 1999; Schaller et al., 1992) (Figure 1). A second member of the family, referred to as PYK2, cell adhesion kinase (CAK)-beta, related adhesion focal tyrosine kinase (RAFTK) or calcium dependent protein tyrosine kinase (CADTK) (Avraham et al., 1995; Lev et al., 1995; Sasaki et al., 1995; Yu et al., 1996) shares signi®cant sequence similarity with FAK. The expression of this FAK-related kinase appears more restricted than that of FAK, being expressed at high levels in brain and lower levels in liver, kidney, spleen, lung and cells of hematopoietic origin (Avraham et al., 1995; Lev et al., 1995; Sasaki et al., 1995). The C-terminal, non-catalytic domain of both FAK and PYK2, termed FRNK (FAK-RelatedNon-Kinase) and PRNK (PYK2 Related NonKinase) respectively, are autonomously expressed in certain cells and may function as negative regulators of their activity (Nolan et al., 1999; Schaller et al., 1993; Xiong et al., 1998; Richardson and Parsons, 1996).
Clustering of integrins leads to the rapid recruitment of FAK to the focal adhesion complex and its concurrent phosphorylation on tyrosine (Parsons and Parsons, 1997). `Activated' FAK is phosphorylated to high stoichiometry on tyrosine 397 (Tyr397), a major site of phosphorylation (Schaller et al., 1994), as well as at several additional sites within the kinase and Cterminal domains (Calalb et al., 1995). Phosphorylation at Tyr397 correlates with increased catalytic activity of FAK (Calalb et al., 1995; Lipfert et al., 1992) and appears important for the tyrosine phosphorylation of focal complex associated proteins such as paxillin and Cas. The phosphorylation on Tyr397 also creates a high anity binding site recognized by the SH2 domain of Src family kinases and leads to the recruitment and activation of Src through the formation of a bipartite kinase complex. (Cobb et al., 1994; Schaller et al., 1994, 1999) (Figure 2). Activation of the FAK-Src complex is central to regulation of downstream signaling pathways that control cell spreading, cell movement and cell survival. Phosphorylation of Tyr397 also appears to be important for the recruitment of other SH2-containing proteins, including the 85 kDa subunit of phosphotidylinositol 3-kinase (PI3 kinase), phospholipase C (PLC)-g and the adapter protein Grb7 (Chen et al., 1996; Chen and Guan, 1994; Han and Guan, 1999). The phosphorylation of Tyr397 as well as Tyr925 creates a binding site for the Grb2-SOS complex (Chen and Guan, 1994; Schlaepfer et al., 1994; Schlaepfer and Hunter, 1996). Finally, FAK phosphorylation at tyrosines 576 and 577 appears important for the maximal adhesion-induced activation of FAK and signaling to downstream eectors (Calalb et al., 1995; Owen et al., 1999). The identi®cation and characterization of FAK binding partners has provided important information about how FAK serves to mediate signaling from adhesion complexes. The targeting of FAK to adhesion complexes is mediated by an approximately 100 amino acid domain within the C-terminus termed the Focal Adhesion Targeting (FAT) region (Figure 1). Se-
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Figure 1 Schematic representation of the organization of the domains of Focal Adhesion Kinase. The N-terminal domain shares similarity with Band 4.1 proteins and directs interactions with integrins and growth factor receptors. The central domain is the catalytic domain. The C-terminal domain contains sites for multiple protein ± protein interactions. SI denotes Site I, an interaction site with the SH3 domain of Cas; SII denotes Site II, a site of interaction with the SH3 domains of GRAF and ASAP. Y397 is the major site of autophosphorylation and a site of interaction with the SH2 domain of Src. FAT denotes the region required for focal adhesion targeting. Paxillin interacts with sequences that overlap the FAT domain. Additional sites of tyrosine and serine phosphorylation are indicated Oncogene
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Figure 2 Schematic representation of the proposed interactions among the proteins in integrin signaling. The large black arrow (top) denoted the proposed interaction between integrin receptors and growth factor receptors. See text for details
quences within the FAT domain are both necessary and sucient to target FAK to adhesion complexes (Hildebrand et al., 1993) and the integrity of this region is essential for FAK signaling (Sieg et al., 1999; Thomas et al., 1999) (Figure 1). The only binding partner for FAT identi®ed to date, is the adhesion complex protein, paxillin. Paxillin binding to FAK is mediated by sequences which signi®cantly overlap the region required for focal adhesion targeting (Hildebrand et al., 1995; Tachibana et al., 1995). Because paxillin has been shown to bind directly to the cytoplasmic domains of integrin receptors (Liu et al., 1999; Schaller et al., 1995) as well as to the focal adhesion protein vinculin, paxillin (or paxillin-like proteins) may function as the `docking partner' for FAK in adhesion complexes. The function of the N-terminal domain of FAK is unclear. The N-terminal domain has been reported to interact in vitro with sequences in the cytoplasmic domain of b-integrin subunits (Schaller et al., 1995); however, a demonstration of a direct interaction between FAK and integrin receptors in vivo is lacking. The N-terminal domain exhibits similarity with Band 4.1 proteins (Girault et al., 1999) although the signi®cance of this homology is unknown. The Nterminal domain has recently been shown to direct the interaction of FAK with the activated forms of the Epidermal Growth Factor (EGF) receptor, although it is not clear whether such interactions are direct or via another protein(s) (Sieg et al., 2000). The C-terminal domain of FAK is rich in protein ± protein interaction sites. A proline-rich sequence designated Site I (Figure 1) provides the major binding motif recognized by the SH3 domain of Cas, a multifunctional linker protein (Harte et al., 1996; O'Neill et Oncogene
al., 2000; Polte and Hanks, 1995). Upon integrin clustering, Cas is localized to adhesion complexes and is phosphorylated on tyrosine (Harte et al., 1996; O'Neill et al., 2000; Petch et al., 1995; Polte and Hanks, 1995). Mutants in FAK lacking the binding site for Cas are compromised in signaling to downstream eectors (see below). A second proline motif within the C-terminal domain, Site II, targets the binding the SH3 domains of two regulators of small GTPases, GRAF, a GAP (GTPase activating protein) for Rho, and ASAPI, a GAP for Arfs 1 and 6 (Randazzo et al., 2000; Taylor et al., 1998, 1999). The role of this binding site in mediating FAK signaling is unclear. To date, mutation of the GRAF/ASAP binding site in FAK has failed to reveal defects in FAK signaling. In addition, alignment of Drosophila and mammalian FAK sequences reveals the conservation of the Cas binding site (Site I) but not the GRAF/ASAP1 binding site (Site II) (Fox et al., 1999). In higher organisms, Site II may have evolved to confer unique signaling to specialized GTPases. Thus binding of GRAF and/or ASAP to FAK may serve to link focal complex signaling with the concerted regulation of small GTP binding proteins in the Rho and Arf families, proteins that clearly play an important function in cytoskeletal reorganization. Recently, four sites of serine phosphorylation have been mapped within the C-terminal domain of FAK (serines 722, 843, 846, and 910). The role of serine phosphorylation in the regulation of FAK function is poorly understood; however, the proximity of several of these phosphorylated serine residues to sites of protein ± protein interactions is provocative (A Ma, A Richardson, E Schaefer and JT Parsons, submitted for publication).
FAK and cell migration JT Parsons et al
Small GTPases and actin dynamics Cell movement is highly dependent upon the dynamic reorganization of the actin cytoskeleton, a process that is tightly coupled to the activation of members of the Rho family of small GTPases. Cdc42 and Rac play a critical role in the formation and organization of cortical actin networks and the protrusion of ®lopodia and lamellipodia (Hall, 1998). Treatment of cells with agents that increase GTP-bound Cdc42 stimulates ®lopodia formation (Kozma et al., 1995), whereas activation of Rac leads to membrane rues and lamellipodia formation (Ridley et al., 1992). Formation of cortical actin networks requires de novo formation of F-actin ®laments, indicating that Cdc42 and Rac integrate signaling pathways leading to actin polymerization. In addition, Rac activation is closely coupled to activation of Cdc42 (Nobes and Hall, 1995), allowing for the coincident and coordinated formation of ®lopodia and lamellipodia that are often concurrently observed at the leading edge in motile cells. Actin polymerization at the leading edge provides the protrusive force required for the extension of lamellipodia and the formation of integrin-containing adhesive complexes (focal complexes) observed during cell movement and spreading (Rottner et al., 1999). Activation of Rho, leads to the formation of actin stress ®bers along with the near-simultaneous occurrence of focal adhesions at their termini. During cell migration, it has been proposed that Cdc42/Rac and Rho signaling are reciprocally controlled, leading to the breakdown of stress ®bers (downregulation of Rho) and the commensurate reorganization of cortical actin networks at the leading edge of the cells as a result of the enhanced activation of the Cdc42/Rac pathway (Burridge, 1999; Horwitz and Parsons, 1999; Rottner et al., 1999). As cell movement progresses, focal adhesions reassemble behind the leading edge presumably re¯ecting the upregulation of Rho, thus providing attachment sites to anchor actin stress ®bers and to support the contractile forces necessary for continued cell movement. Cortical actin polymerization is regulated by Cdc42 and Rac via their interaction with members of the Wiskott-Aldrich Syndrome protein (WASp)/Scar1 superfamily (Mullins, 2000). Interaction of Cdc42/Rac with WASp/Scar proteins unmasks a C-terminal acidic region which mediates binding of WASp/Scar to the Arp2/3 complex (Machesky and Gould, 1999). Arp2/3, a complex comprised of seven proteins (Machesky and Gould, 1999), binds to the sides of preexisting actin ®laments and stimulates new ®lament formation to create branched actin networks (Mullins et al., 1998). Arp2/3-induced actin nucleation and polymerization is greatly enhanced by binding of WASp-family acidic carboxyl-terminal domains to the Arp2/3 complex (Machesky et al., 1999), and thus provides a molecular link for Cdc42 and Rac induction of cortical actin polymerization (Machesky et al., 1999; Rohatgi et al., 1999). Rho regulates the further organization of actin into bundles and the production focal adhesions (Burridge and Chrzanowska-Wodnicka, 1996). Rho also acts to promote tension, via its regulation of Rho kinase and myosin phosphatase and the subsequent regulation of myosin light chain (MLC) phosphorylation. Rho
activation of Rho kinase inhibits the myosin phosphatase thus maintaining MLCs in a highly phosphorylated (contractile) state. The resultant contractile forces are important in the organization of the actin ®laments. The regulation of Rac and Rho are highly interconnected and may vary spatially and temporally within the cell (Rottner et al., 1999). A key step in cell migration is the reciprocal (indeed cyclical) regulation of Rac, which appears active at the cell edge where new protrusions and adhesions are forming, and Rho, which appears to generate tension and stabilize adhesions more centrally throughout the cell.
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Role for FAK and binding partners in cell migration Several lines of evidence implicate FAK in the regulation of cell migration. First, FAK de®cient cells migrate poorly in response to chemotactic and haptotactic signals, although they form large, prominent focal adhesions (Ilic et al., 1995; Owen et al., 1999; Sieg et al., 1999). Second, over expression of FRNK, the autonomously expressed C-terminal domain of FAK, blocks cell spreading as well as chemotactic and haptotactic migration (Richardson et al., 1997; Sieg et al., 1999). Finally, over expression of FAK in Chinese hamster ovary (CHO) cells enhances cell migration (Cary et al., 1996). The reconstitution of FAK de®cient cells with wild-type FAK restores cell migration, whereas reintroduction of FAK mutants de®cient for Src family kinase binding (Y397 mutation), lacking kinase activity or de®cient for Cas binding (Site I mutation) fails to restore full haptotactic cell migration (Sieg et al., 1999; Cary et al., 1998). Interestingly, direct binding of paxillin does not appear to be required to reconstitute FAK directed cell migration. These experiments clearly support a role for FAK in the regulation of signaling events required for cell migration and further point to the importance of Cas and Src family kinase interactions with FAK for ecient downstream signaling. This latter conclusion is further supported by the observation that cells de®cient for Src, Yes and Fyn or cells de®cient for Cas display reduced haptotactic migration (Honda et al., 1999; Klinghoer et al., 1999). Analysis of the signaling pathways mediated by FAK binding partners provides insights into how FAK might contribute to the regulation of cell migration. The FAK binding partner Cas appears to function as an important `molecular switch' for the induction of migration signals via its binding to the SH2/SH3adapter protein Crk (Vuori et al., 1996). The expression of CAS or Crk is sucient to promote cell migration on several matrix substrates. In addition, enhanced cell migration induced by Cas is dependent upon CAS tyrosine phosphorylation and formation of Cas-Crk complexes (Klemke et al., 1998). Cytokinestimulated cell migration is also blocked by dominant negative forms of Cas or Crk, e.g., expression of CAS lacking the Crk binding site or by expression of Crk containing a mutant SH2 domain (Klemke et al., 1998). These data indicate that chemotactic migration induced by some growth factor receptors may signal in part through Cas. Enhanced haptotactic migration in response to Cas/Crk expression is blocked by dominant-negative Rac but not dominant-negative forms of Oncogene
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Ras. These data indicate that FAK/Src-dependent activation of the Cas/Crk signaling complex is a central feature of both matrix and growth factor induced cell migration. Furthermore, the inhibition of cell migration by expression of dominant-negative forms of Rac provides evidence for Rac being an important downstream eector of the FAK/Src-Cas/ Crk pathway. DOCK180 is the human counterpart of the Drosophila melanogaster and Caenorhabditis elegans genes mbc and ced-5, genes implicated in the regulation of phagacytic and cell migratory events (Erickson et al., 1997; Wu and Horvitz, 1998). DOCK180 appears to be important in the regulation of signals from Cas/ Crk complexes. Spreading of cells on ®bronectin, leads to an increase in Cas/Crk complex formation and a concomitant increase in Crk/DOCK180 complexes. Furthermore, co-expression of DOCK180 along with Cas and Crk promotes membrane ruing and accumulation of DOCK180/Cas/Crk complexes in focal adhesions (Kiyokawa et al., 1998b). Although DOCK180 shows no sequence similarity to guanine nucleotide exchange factors (GEFs), over expression of DOCK180 leads to an increase in GTP-bound Rac (Kiyokawa et al., 1998a). Dominant-negative Rac suppresses DOCK180 induced membrane ruing as well as DOCK180 induced cell migration (Cheresh et al., 1999), further pointing to Rac as an important downstream target of the FAK/Src-Cas/CrkDOCK180 signaling pathway (Figure 2). A possible role for this pathway might be the activation of Rac in response to integrin engagement (focal complex formation) at the leading edge of the cell, thus providing additional protrusive activity and the extension of new lamellipodia via the Rac-induced activation of the Arp2/3 cortical actin polymerization. Therefore, in haptotactic migration where migratory signals are generated by engagement of integrin receptors with ECM proteins, the FAK/Src-Cas/CrkDOCK signaling may represent a dominant pathway for the activation of Rac and cell migration. Paxillin appears to regulate at least two distinct pathways which converge to activate Rac. Cells spread on ®bronectin or other ECM proteins exhibit an increase in the phosphorylation of paxillin on tyrosines 31 and 118. Phosphorylation of these residues enhances Crk binding to paxillin and formation of paxillin/Crk complexes (Schaller and Parsons, 1995). Recent experiments show that bladder tumor cell migration on collagen is impaired upon over expression of a mutant form of paxillin lacking tyrosines 31 and 118, whereas cell adhesion and spreading are not aected. Over expression of wild-type paxillin or Crk is able to overcome the migration de®cient phenotype (Petit et al., 2000). These data suggest that activation of DOCK180 signaling to Rac may play a central role in the paxillin-Crk signaling pathway, as it does in the Cas-Crk pathway (Figure 2). Paxillin has also been shown to bind to a second complex of proteins with unique signaling properties. Paxillin contains ®ve copies of a leucine rich repeat (LD motif) within its N-terminal region. The LD motifs of paxillin are contained within a region that directs binding of paxillin to FAK, vinculin and the E6 oncoprotein from papillomavirus (Turner et al., 1999; Vande Pol et al., 1998). LD4 has recently been shown
to bind a complex of proteins containing PAK (p21 activated kinase), the SH2/SH3-adapter protein Nck, two members of the Cdc42/Rac GEF family (PIX/ COOL) and a multi-domain ARF-GAP protein, PKL (paxillin-kinase linker) (Bagrodia et al., 1999; Bagrodia and Cerione, 1999; Turner et al., 1999). PKL binds paxillin and PIX directly in vitro suggesting that paxillin serves to recruit both PKL and PIX to adhesion structures. In support of this role of paxillin is the observation that GFP-tagged PKL localizes to focal adhesions/complexes in CHO cells. Furthermore, over expression of a paxillin LD4 deletion mutant inhibits lamellipodia formation in response to growth factor stimulation and signi®cantly reduces cell migration into a wound (Turner et al., 1999). The PIX/ COOL family of proteins was originally identi®ed as regulators of the Cdc42/Rac activated kinase, PAK because of their ability to bind and activate these kinases (Manser et al., 1998). PIX proteins were originally reported to exhibit GEF activity for Rac and Cdc42, although this property of PIX proteins has recently been questioned, raising speculation that PIX/ COOL proteins may activate PAK by binding the GTP form of Cdc42/Rac rather than directly activating the formation of the GTP form of the small GTPases (Bagrodia et al., 1999). An intriguing aspect of paxillin signaling is that it implicates paxillin in both the regulation of Rac and Pak, a well recognized regulator of the actin cytoskeleton. FAK signaling to protein and lipid kinases PAK kinases appear to be important regulators of cytoskeletal rearrangements and lamellipodia formation (Sells et al., 1999). Integrin dependent adhesion leads to the rapid activation of PAK in a Cdc42/Rac dependent manner (del Pozo et al., 2000). Expression of dominant-negative forms of PAK (de®cient in GTPase binding and catalytic activity) in endothelial cells signi®cantly inhibits cell migration, increases the number of stress ®bers and size of focal adhesions but has little eect on lamellipodia formation (Kiosses et al., 1999). In NIH3T3 cells, over expression of wildtype or constitutively active PAK stimulates the formation of large polarized lamellipodia at the leading edge of cells (Sells et al., 1999). Interestingly, in these cells, over expression of catalytically inactive PAK leads to apolar formation of lamellipodia and reduction of directed motility on collagen substrates. In both endothelial cells and ®broblasts, over expression of activated PAK leads to an increase in myosin light chain phosphorylation. These studies support a model in which recruitment and activation of PAK at the site of integrin engagement may be important in the formation of stable adhesions at the leading edge of the cell and perhaps the modulation of actin-myosin contraction required for the transition of focal complexes to focal adhesions. A role for ERK activation in cell adhesion and migration on ECM substrates is poorly understood. Integrin engagement or cell spreading of ECM substrates is sucient to activate the ERK signaling pathway (Chen et al., 1994; Renshaw et al., 1996; Zhu and Assoian, 1995) and target the localization of ERK to peripheral adhesion complexes (Fincham et al.,
FAK and cell migration JT Parsons et al
2000). The adhesion dependent activation of Erk is notably weaker albeit sustained compared to the robust and transient activation of ERK observed upon growth factor receptor ligation. The sustained activation of ERK presumably re¯ects the prolonged and timedependent formation of multiple adhesions during the process of cell spreading (Lin et al., 1997; Zhu and Assoian, 1995). In some cell types, inhibition of MEK activation with the MEK inhibitor U0126 blocks cell spreading and haptotactic cell migration but not cell attachment, consistent with a requirement for ERK activation in the formation of stable adhesion structures (Cheresh et al., 1999; Fincham et al., 2000). How ERK activation by integrins is regulated is unclear. Three seemly independent pathways have been described. Phosphorylation of FAK on Tyr925 has been reported to recruit the SH2 domain of Grb2, resulting in the activation of the SOS-Ras-Raf-MEKERK pathway (Schlaepfer et al., 1994; Schlaepfer and Hunter, 1996). Antibody mediated clustering of beta-1 or alpha-v integrins stimulates ligand independent activation of the EGF receptor and the subsequent activation of the Ras-Raf-MEK-ERK signaling pathway (Moro et al., 1998). However integrin dependent activation of EGF receptor signaling is clearly dependent upon the threshold expression of approximately 5000 EGF receptors on the cell surface. Finally, certain alpha subunits of the integrin receptors signal directly via Shc to the Grb2/SOS pathway. Upon integrin engagement, the Src family kinase, Fyn links to the cytoplasmic domain of the receptor through a process involving caveolin-1. Activated Fyn binds and phosphorylates Shc, leading to the recruitment of Grb2 and activation of the Ras-Raf-MEK-ERK pathway. It is unclear how the balance of integrin signaling to ERK is regulated using these three pathways. Nonetheless, it is certainly possible that dierent ECM environments, or variations in the surface expression of growth factor receptors could contribute signi®cantly to ERK signaling, potentiating cell movement. ERK activation also appears important in the regulation of actin-myosin mediated contraction. ERK directly phosphorylates and activates MLCK, (MLC kinase) thereby stimulating the phosphorylation of myosin light chains (Klemke et al., 1997). The increased contractility induced by phosphorylation of myosin light chains is important for several key processes in cell migration including the formation of both peripheral adhesion complexes and focal adhesions (Burridge, 1999; Burridge and ChrzanowskaWodnicka, 1996; Horwitz and Parsons, 1999). Phosphorylation of other focal adhesion proteins may also be important for regulation of adhesion complex formation and/or turnover. For example, the FAK associated Rho-GAP, GRAF is an in vitro and in vivo substrate for ERK although the physiological consequences of ERK phosphorylation remain to be established (Taylor et al., 1998). In EL4 cells, paxillin is phosphorylated at several sites by the activation of ERK although again, the physiological signi®cance of these phosphorylations remains to be determined (Ku and Meier, 2000). Phosphorylation of the LIM
domains of paxillin appear important for the localization of paxillin to focal adhesions (Brown et al., 1998). Undoubtedly other focal adhesion proteins will be unmasked as substrates for activated ERK or other related protein kinases. Integrin activation of PI3 kinase is an important mediator of a number of signaling pathways required for cell migration. The direct activation of PI3 kinase by FAK is indicated by the demonstration that the p85 subunit of PI3 kinase directly associates with Tyr397 upon integrin ligation (Chen and Guan, 1994). Furthermore, inhibitors of PI3 kinase inhibit FAK promoted migration of CHO cells in a dose dependent manner. A mutant of FAK capable of binding Src but not PI3 kinase fails to promote migration of CHO cells when over expressed, indicating that FAK plays a role in the integrin-dependent activation of PI3 kinase. Given the central role of lipid kinases in regulating actin cytoskeletal organization and adhesion assembly, it is likely that other integrin-stimulated pathways will converge to activate PI3 kinase.
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Concluding remarks Cell migration is a dynamic process requiring the integration of multiple signaling pathways. Migration of cells on ECM proteins requires the reiterative process of lamellipodia extension, integrin mediated adhesion, focal complex formation and the transition of focal complexes to focal adhesions, and ®nally the release of adhesion complexes at the rear of the cell. Protein tyrosine kinases, and FAK in particular, appear to play a central role in the dynamic regulation of cell adhesion structures. As discussed above, FAK is likely to play a direct role in regulating signals to the small GTPase Rac via interactions with Cas/Crk and Paxillin/Crk and paxillin/PLK/PIX and to PI3 kinase by its direct binding to FAK. Both Rac and PI3 kinase are intimately involved in the regulation of cortical actin and lamellipodia and are essential for the process of cell migration. A number of very important questions remain unanswered. Little is known about the mechanisms that regulate the recruitment and organization of components of focal complexes and focal adhesions. Are the signals emanating from focal complexes dierent from those provided by focal adhesions? How does tyrosine phosphorylation of FAK, Cas and paxillin contribute to the dynamic nature of adhesion complexes ± are other phosphorylations/kinases important? Finally, how do the signal transduction events studied in cultured cells relate to signaling in the complex organization of individual tissues? Much remains to be done!
Acknowledgments We wish to thank our many colleagues who have patiently explained the vagaries of integrin biology to us over the years. We thank Lynn McCutcheon and Cli Martin for editorial and artistic help. JT Parsons acknowledges the support of NIH grants, CA40042 and CA40042.
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References Avraham S, London R, Fu Y, Ota S, Hiregowdara D, Li J, Jiang S, Pasztor LM, White RA, Groopman JE and Avraham H. (1995). J. Biol. Chem., 270, 27742 ± 27751. Bagrodia S, Bailey D, Lenard Z, Hart M, Guan JL, Premont RT, Taylor SI and Cerione RA. (1999). J. Biol. Chem., 274, 22393 ± 22400. Bagrodia S and Cerione RA. (1999). Trends. Cell. Biol., 9, 350 ± 355. Brown MC, Perrotta JA and Turner CE. (1998). Mol. Biol. Cell, 9, 1803 ± 1816. Burridge K. (1999). Science, 283, 2028 ± 2029. Burridge K and Chrzanowska-Wodnicka M. (1996). Annu. Rev. Cell. Dev. Biol., 12, 463 ± 518. Burridge K, Fath K, Kelly T, Nuckolls G and Turner C. (1988). Annu. Rev. Cell. Biol., 4, 487 ± 525. Calalb MB, Polte TR and Hanks SK. (1995). Mol. Cell. Biol., 15, 954 ± 963. Calderwood DA, Shattil SJ and Ginsberg MH. (2000). J. Biol. Chem., 275, 22607 ± 22610. Cary LA, Chang JF and Guan JL. (1996). J. Cell. Sci., 109, 1787 ± 1794. Cary LA, Han DC, Polte TR, Hanks SK and Guan JL. (1998). J. Cell. Biol., 140, 211 ± 221. Chen HC, Appeddu PA, Isoda H and Guan JL. (1996). J. Biol. Chem., 271, 26329 ± 26334. Chen HC and Guan JL. (1994). Proc. Natl. Acad. Sci. USA, 91, 10148 ± 10152. Chen Q, Kinch MS, Lin TH, Burridge K and Juliano RL. (1994). J. Biol. Chem., 269, 26602 ± 26605. Cheresh DA, Leng J and Klemke RL. (1999). J. Cell. Biol., 146, 1107 ± 1116. Cobb BS, Schaller MD, Leu TH and Parsons JT. (1994). Mol. Cell. Biol., 14, 147 ± 155. Craig SW and Johnson RP. (1996). Curr. Opin. Cell. Biol., 8, 74 ± 85. del Pozo MA, Price LS, Alderson NB, Ren XD and Schwartz MA. (2000). EMBO J., 19, 2008 ± 2014. Erickson MR, Galletta BJ and Abmayr SM. (1997). J. Cell. Biol., 138, 589 ± 603. Fincham VJ, James M, Frame MC and Winder SJ. (2000). EMBO J., 19, 2911 ± 2923. Fox GL, Rebay I and Hynes RO. (1999). Proc. Natl. Acad. Sci. USA, 96, 14978 ± 14983. Girault JA, Labesse G, Mornon JP and Callebaut I. (1999). Trends. Biochem. Sci., 24, 54 ± 57. Guan JL, Trevithick JE and Hynes RO. (1991). Cell. Regul., 2, 951 ± 964. Hall A. (1998). Science, 279, 509 ± 514. Han DC and Guan JL. (1999). J. Biol. Chem., 274, 24425 ± 24430. Hanks SK, Calalb MB, Harper MC and Patel SK. (1992). Proc. Natl. Acad. Sci. USA, 89, 8487 ± 8491. Harte MT, Hildebrand JD, Burnham MR, Bouton AH and Parsons JT. (1996). J. Biol. Chem., 271, 13649 ± 13655. Hildebrand JD, Schaller MD and Parsons JT. (1993). J. Cell. Biol., 123, 993 ± 1005. Hildebrand JD, Schaller MD and Parsons JT. (1995). Mol. Biol. Cell, 6, 637 ± 647. Honda H, Nakamoto T, Sakai R and Hirai H. (1999). Biochem. Biophys. Res. Commun., 262, 25 ± 30. Horwitz AR and Parsons JT. (1999). Science, 286, 1102 ± 1103. Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M and Yamamoto T. (1995). Nature, 377, 539 ± 544. Kiosses WB, Daniels RH, Otey C, Bokoch GM and Schwartz MA. (1999). J. Cell. Biol., 147, 831 ± 844. Kiyokawa E, Hashimoto Y, Kobayashi S, Sugimura H, Kurata T and Matsuda M. (1998a). Genes. Dev., 12, 3331 ± 3336.
Oncogene
Kiyokawa E, Hashimoto Y, Kurata T, Sugimura H and Matsuda M. (1998b). J. Biol. Chem., 273, 24479 ± 24484. Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P and Cheresh DA. (1997). J. Cell. Biol., 137, 481 ± 492. Klemke RL, Leng J, Molander R, Brooks PC, Vuori K and Cheresh DA. (1998). J. Cell. Biol., 140, 961 ± 972. Klinghoer RA, Sachsenmaier C, Cooper JA and Soriano P. (1999). EMBO J., 18, 2459 ± 2471. Kornberg L, Earp HS, Parsons JT, Schaller M and Juliano RL. (1992). J. Biol. Chem., 267, 23439 ± 23442. Kozma R, Ahmed S, Best A and Lim L. (1995). Mol. Cell. Biol., 15, 1942 ± 1952. Ku H and Meier KE. (2000). J. Biol. Chem., 275, 11333 ± 11340. Lauenburger DA and Horwitz AF. (1996). Cell, 84, 359 ± 369. Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B and Schlessinger J. (1995). Nature, 376, 737 ± 745. Lin TH, Aplin AE, Shen Y, Chen Q, Schaller M, Romer L, Aukhil I and Juliano RL. (1997). J. Cell. Biol., 136, 1385 ± 1395. Lipfert L, Haimovich B, Schaller MD, Cobb BS, Parsons JT and Brugge JS. (1992). J. Cell. Biol., 119, 905 ± 912. Liu S, Thomas SM, Woodside DG, Rose DM, Kiosses WB, Pfa M and Ginsberg MH. (1999). Nature, 402, 676 ± 681. Machesky LM and Gould KL. (1999). Curr. Opin. Cell. Biol., 11, 117 ± 121. Machesky LM, Mullins RD, Higgs HN, Kaiser DA, Blanchoin L, May RC, Hall ME and Pollard TD. (1999). Proc. Natl. Acad. Sci. USA, 96, 3739 ± 3744. Manser E, Loo TH, Koh CG, Zhao ZS, Chen XQ, Tan L, Tan I, Leung T and Lim L. (1998). Mol. Cell., 1, 183 ± 192. Moro L, Venturino M, Bozzo C, Silengo L, Altruda F, Beguinot L, Tarone G and De®lippi P. (1998). EMBO J., 17, 6622 ± 6632. Mullins RD. (2000). Curr. Opin. Cell. Biol., 12, 91 ± 96. Mullins RD, Heuser JA and Pollard TD. (1998). Proc. Natl. Acad. Sci. USA, 95, 6181 ± 6186. Nobes CD and Hall A. (1995). Cell., 81, 53 ± 62. Nolan K, Lacoste J and Parsons JT. (1999). Mol. Cell. Biol., 19, 6120 ± 6129. O'Neill GM, Fashena SJ and Golemis EA. (2000). Trends Cell. Biol., 10, 111 ± 119. Owen JD, Ruest PJ, Fry DW and Hanks SK. (1999). Mol. Cell. Biol., 19, 4806 ± 4818. Palmer RH, Fessler LI, Edeen PT, Madigan SJ, McKeown M and Hunter T. (1999). J. Biol. Chem., 274, 35621 ± 35629. Parsons JT and Parsons SJ. (1997). Curr. Opin. Cell. Biol., 9, 187 ± 192. Petch LA, Bockholt SM, Bouton A, Parsons JT and Burridge K. (1995). J. Cell. Sci., 108, 1371 ± 1379. Petit V, Boyer B, Lentz D, Turner CE, Thiery JP and Valles AM. (2000). J. Cell. Biol., 148, 957 ± 970. Polte TR and Hanks SK. (1995). Proc. Natl. Acad. Sci. USA, 92, 10678 ± 10682. Randazzo PA, Andrade J, Miura K, Brown MT, Long YQ, Stauer S, Roller P and Cooper JA. (2000). Proc. Natl. Acad. Sci. USA, 97, 4011 ± 4016. Renshaw MW, Toksoz D and Schwartz MA. (1996). J. Biol. Chem., 271, 21691 ± 21694. Richardson A, Malik RK, Hildebrand JD and Parsons JT. (1997). Mol. Cell. Biol., 17, 6906 ± 6914. Richardson A and Parsons T. (1996). Nature, 380, 538 ± 540. Ridley AJ, Paterson HF, Johnston CL, Diekmann D and Hall A. (1992). Cell., 70, 401 ± 410. Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T and Kirschner MW. (1999). Cell., 97, 221 ± 231.
FAK and cell migration JT Parsons et al
Rottner K, Hall A and Small JV. (1999). Curr. Biol., 9, 640 ± 648. Sasaki H, Nagura K, Ishino M, Tobioka H, Kotani K and Sasaki T. (1995). J. Biol. Chem., 270, 21206 ± 21219. Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB and Parsons JT. (1992). Proc. Natl. Acad. Sci. USA, 89, 5192 ± 5196. Schaller MD, Borgman CA and Parsons JT. (1993). Mol. Cell. Biol., 13, 785 ± 791. Schaller MD, Hildebrand JD and Parsons JT. (1999). Mol. Biol. Cell., 10, 3489 ± 3505. Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR and Parsons JT. (1994). Mol. Cell. Biol., 14, 1680 ± 1688. Schaller MD, Otey CA, Hildebrand JD and Parsons JT. (1995). J. Cell. Biol., 130, 1181 ± 1187. Schaller MD and Parsons JT. (1995). Mol. Cell. Biol., 15, 2635 ± 2645. Schlaepfer DD, Hanks SK, Hunter T and van der Geer P. (1994). Nature, 372, 786 ± 791. Schlaepfer DD and Hunter T. (1996). Mol. Cell. Biol., 16, 5623 ± 5633. Schwartz MA, Schaller MD and Ginsberg MH. (1995). Annu. Rev. Cell. Dev. Biol., 11, 549 ± 599. Sells MA, Boyd JT and Cherno J. (1999). J. Cell. Biol., 145, 837 ± 849. Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH and Schlaepfer DD. (2000). Nat. Cell. Biol., 2, 249 ± 256. Sieg DJ, Hauck CR and Schlaepfer DD. (1999). J. Cell. Sci., 112, 2677 ± 2691.
Tachibana K, Sato T, D'Avirro N and Morimoto C. (1995). J. Exp. Med., 182, 1089 ± 1099. Taylor JM, Hildebrand JD, Mack CP, Cox ME and Parsons JT. (1998). J. Biol. Chem., 273, 8063 ± 8070. Taylor JM, Macklem MM and Parsons JT. (1999). J. Cell. Sci., 112, 231 ± 242. Thomas JW, Cooley MA, Broome JM, Salgia R, Grin JD, Lombardo CR and Schaller MD. (1999). J. Biol. Chem., 274, 36684 ± 36692. Turner CE, Brown MC, Perrotta JA, Riedy MC, Nikolopoulos SN, McDonald AR, Bagrodia S, Thomas S and Leventhal PS. (1999). J. Cell. Biol., 145, 851 ± 863. Vande Pol SB, Brown MC and Turner CE. (1998). Oncogene, 16, 43 ± 52. Vuori K, Hirai H, Aizawa S and Ruoslahti E. (1996). Mol. Cell. Biol., 16, 2606 ± 2613. Wu YC and Horvitz HR. (1998). Nature, 392, 501 ± 504. Xiong WC, Macklem M and Parsons JT. (1998). J. Cell. Sci., 111, 1981 ± 1991. Yu H, Li X, Marchetto GS, Dy R, Hunter D, Calvo B, Dawson TL, Wilm M, Anderegg RJ, Graves LM and Earp HS. (1996). J. Biol. Chem., 271, 29993 ± 29998. Zamir E, Katz BZ, Aota S, Yamada KM, Geiger B and Kam Z. (1999). J. Cell. Sci., 112, 1655 ± 1669. Zamir E, Katz M, Posen Y, Erez N, Yamada KM, Katz BZ, Lin S, Lin DC, Bershadsky A, Kam Z and Geiger B. (2000). Nat. Cell. Biol., 2, 191 ± 196. Zhu X and Assoian RK. (1995). Mol. Biol. Cell., 6, 273 ± 282.
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Oncogene
