FOCAL ADHESION KINASE: IN COMMAND AND CONTROL OF CELL MOTILITY

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1 FOCAL ADHESION KINASE: IN COMMAND AND CONTROL OF CELL MOTILITY Satyajit K. Mitra, Daniel A. Hanson and David D. Schlaepfer Abstract A central question in cell biology is how membrane-spanning receptors transmit extracellular signals inside cells to modulate cell adhesion and motility. Focal adhesion kinase () is a crucial signalling component that is activated by numerous stimuli and functions as a biosensor or integrator to control cell motility. Through multifaceted and diverse molecular connections, can influence the cytoskeleton, structures of cell adhesion sites and membrane protrusions to regulate cell movement. INTEGRINS A large family of heterodimeric transmembrane proteins that function as receptors for celladhesion molecules. EXTRACELLULAR MATRIX (ECM). The complex, multimolecular material that surrounds cells. The ECM comprises a scaffold on which tissues are organized, it provides cellular microenvironments and it regulates various cellular functions. The Scripps Research Institute, Department of Immunology, IMM North Torrey ines Road, La Jolla, California 92037, USA. Correspondence to D.D.S. dschlaep@scripps.edu doi: /nrm1549 Cell migration is a coordinated process that involves rapid changes in the dynamics of actin filaments, together with the formation and disassembly of cell adhesion sites 1.A complex interplay between the actin cytoskeleton and cell adhesion sites leads to the generation of membrane protrusions and traction forces 2. External stimuli that control cell migration are transduced into intracellular biochemical signals through the interactions of transmembrane INTEGRINS that bind to EXTRACELLULAR MATRIX (ECM) proteins, growth factors that bind to their cognate cell-surface receptors, or mechanical stimuli such as shear stress that promote deformation of the actin cytoskeleton. For a cell to process these different environmental motility-promoting stimuli correctly, there must be essential intracellular signalling proteins that function as integrators that is, proteins that are stimulated by multiple extracellular inputs and that function to regulate multiple signalling pathway outputs 3. Here, we describe the unique molecular connections of focal adhesion kinase () that allow this tyrosine kinase to function as an important receptor-proximal regulator of cell shape, adhesion and motility. The complexities of was independently identified in 1992 by Steve Hanks, Jun-Lin Guan and Michael Schaller as a substrate of the viral Src oncogene and, in normal cells, as a highly tyrosine-phosphorylated protein that localized to integrin-enriched cell adhesion sites that are known as focal contacts (BOX 1).Focal contacts are formed at ECM integrin junctions that bring together cytoskeletal and signalling proteins during the processes of cell adhesion, spreading and migration. Early studies found that could be activated by either ECM or growth factors, and that tyrosine phosphorylation of was a rapid event that was associated with the formation of focal contacts 4.Subsequent studies using knockout mice revealed that null mutation of resulted in defective developmental morphogenesis 5.As -null fibroblasts show excessive, rather than decreased (as was initially predicted), formation of focal contacts, signalling has been associated with the disassembly of integrin-based adhesion sites 6.The loss of expression also disrupts microtubule polarization within cells 7, and this phenotype, as well as the defect in focal contact turnover, has been linked to the -mediated regulation of RHO-FAMILY GTases in cells 8.Rho-family GTases are molecular switches within cells, which control the formation and disassembly of actin cytoskeletal structures (STRESS FIBRES, LAMELLIODIA and filopodia) and that function to provide the molecular framework that supports directed cell motility. In both normal and transformed cells, signalling can promote increased cell motility. The genomic designation of human is protein-tyrosine kinase-2 56 JANUARY 2005 VOLUME 6

2 Box 1 Molecular architecture of focal contacts lasma membrane axillin Talin Integrins α-actinin Src Actin stress fibres α-actinin Vinculin Zyxin p130 Cas p130 Cas α-actinin α-actinin Src Vinculin Vinculin axillin Talin Focal contact proteins many studies have shown that inhibition blocks the response to cell motility cues 18,recent and provocative studies have shown that inhibition of expression or activity resulted in increased carcinoma cell migration through the dissolution of N-cadherin-mediated cell cell contacts in HeLa CELLS 19. It is possible that this latter observation might be a cell-type-specific signalling event. However, we speculate that the ability of to promote both the maturation and turnover of focal contacts is related to its role as both a signalling kinase and as an adaptor/scaffold protein, which places in a position to modulate various intracellular signalling pathways (FIG. 1). Namely, it is the association of with both activators and/or inhibitors of various small GTase proteins (Rho, Rac, Cdc42 and Ras) that enables changes in activity to be connected to alterations in the polymerization or stabilization of actin and microtubule filaments. Additionally, because migrating cells experience changes in forces through integrin contacts that link the ECM with the cytoskeleton, is important in the sensing of mechanical forces that are either generated internally or exerted on cells 20. activation is therefore involved in modulating corrective cell responses to environmental stimuli. does this through signal-mediated effects on actin polymerization, the assembly or disassembly of focal contacts, and the regulation of protease activation or secretion 16,21,22. The extracellular matrix, integrins (α- and β-transmembrane heterodimeric proteins) and the cell cytoskeleton interact at sites called focal contacts. Focal contacts are dynamic groups of structural and regulatory proteins that transduce external signals to the cell interior and can also relay intracellular signals to generate an activated integrin state at the cell surface 113.The integrin-binding proteins paxillin and talin recruit focal adhesion kinase () and vinculin to focal contacts (see figure). α-actinin is a cytoskeletal protein that is phosphorylated by, binds to vinculin and crosslinks actomyosin stress fibres and tethers them to focal contacts. Zyxin is an α-actinin- and stress-fibrebinding protein that is present in mature contacts. Although the aforementioned proteins are found in most focal contacts, the membrane-associated protein tyrosine kinase Src and the ADATOR ROTEIN p130cas associate with focal contacts following integrin clustering. Integrin-mediated activation is mediated in part by matrix binding or by force-dependent changes in cytoskeletal linkages. Several other proteins such as extracellular signal-regulated kinase 2 (ERK2) and calpain are known to be transiently present at focal contacts (not shown). The composition of a focal contact is therefore constantly varying depending on external cues and cellular responses. RHO-FAMILY GTases A subfamily of small (~21 kda) GT-binding proteins that are related to Ras and that regulate the cytoskeleton. The nucleotide-bound state is regulated by GTase-activating proteins, which catalyse hydrolysis of the bound GT, and guanine nucleotideexchange factors, which catalyse GD GT exchange. α β β Extracellular matrix (TK2) and it is located at human chromosome 8q24term, a locus that is subject to amplification in human cancer cells 9.Furthermore, elevated levels of TK2 mrna have been found in studies of human carcinoma tumours and in acute lymphoblastic leukaemias, as detected by large-scale gene expression profiling 10,11. protein expression is elevated in many highly malignant human cancers 12, and studies have shown that signalling can promote changes in cell shape 13,14 and the formation of podosomes or invadopodia 15, which leads to an invasive cell phenotype 16,17.Whereas α The FERM and FAT domains of is a ubiquitously expressed 125-kDa protein tyrosine kinase that is composed of an N-terminal FERM (protein 4.1, ezrin, radixin and moesin homology) domain, a central kinase domain, proline-rich regions and a C-terminal focal-adhesion targeting (FAT) domain (FIG. 2). The FERM domain of facilitates a signalling linkage from receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (DGFR) 23.In analysing cell-motility-promoting signals that are initiated by G-ROTEIN-COULED RECETORS (GCRs), overexpression of the FERM domain blocked activation and resulted in the inhibition of G-protein-stimulated cell migration 24.It is the FERM domain that can bind to and promote the integrin- and -mediated activation of other non-receptor tyrosine kinases such as ETK 25.Additionally, actin- and membrane-associated adaptor proteins such as ezrin can bind to the FERM domain and facilitate increased activation in an integrin-independent manner 26. How the FERM domain associates with various targets is an active area of research. can become post-translationally modified by the covalent addition of a small ubiquitin-related modifier (SUMO) at the ε-amino position of Lys152 (REF. 27). In most instances, sumoylation is associated with the nuclear import of proteins and, correspondingly, sumoylated was enriched in the nuclear fraction of cells 27.Although blocking nuclear export using leptomycin B promotes the nuclear accumulation of, and exogenous expression of the FERM domain exhibits strong NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 6 JANUARY

3 Extracellular matrix Integrins α β Growth factor receptors directly to the cytoplasmic tails of integrins 33,accumulated evidence supports an indirect association of with integrins through binding to integrinassociated proteins such as paxillin and talin 18.The FAT domain also binds directly to an activator of Rho-family GTases that is known as p190 RhoGEF, and -mediated tyrosine phosphorylation of p190 RhoGEF might be a direct link to RhoA activation 34. Focal contacts Figure 1 Focal adhesion kinase integrates signals to promote cell migration. Focal adhesion kinase () is activated by growth factors and integrins during migration, and functions as a receptor-proximal regulator of cell motility. At contacts between cells and the extracellular matrix, functions as an adaptor protein to recruit other focal contact proteins or their regulators, which affects the assembly or disassembly of focal contacts. activity and downstream signalling can promote changes in actin and microtubule structures, and signalling can affect the formation and disassembly of cell cell (cadherin-based) contacts. The Rho-family GTases (RhoA, Rac and Cdc42) direct local actin assembly into stress fibres, lamellipodia and filopodia, respectively. can influence the activity of Rho-family GTases through a direct interaction with, or phosphorylation of, protein activators or inhibitors of Rho GTases. RhoA can also influence the stability of microtubules through its effector Diaphanous (mdia). STRESS FIBRES Also termed actinmicrofilament bundles, these are bundles of parallel filaments that contain F-actin and other contractile molecules, and often stretch between cell attachments as if under stress. LAMELLIODIA Broad, flat protrusions at the leading edge of a moving cell that are enriched with a branched network of actin filaments. HeLa CELLS An established tissue-culture strain of human epidermoid carcinoma cells, containing chromosomes per cell. These cells were originally derived from tissue taken from a patient named Henrietta Lacks in Assembly Disassembly Microtubule stabilization? Rho-family GTases Cadherins mdia RhoA Rac Cdc42 Cell migration Stress fibres Lamellipodia Filopodia nuclear localization 28, it is not known whether these events are dependent on sumoylation. As sumoylated showed elevated activity 27, and signalling has been linked to enhanced gene transcription 29 and cellcycle progression 30, it is possible that sumoylation of might facilitate a direct signalling route between focal contacts and the nucleus. The C-terminal domain of contains two proline-rich regions that function as binding sites for SRC-HOMOLOGY (SH)3-DOMAIN-containing proteins (FIG. 2). SH3-domain-mediated binding of the adaptor protein p130cas to is important in promoting cell migration through the coordinated activation of Rac at membrane extensions 31,32.The SH3-mediated binding of other proteins, such as GRAF (GTase regulator associated with ) and ASA1 (Arf GTase-ACTIVATING ROTEIN (GA) containing SH3, ankyrin repeat and pleckstrin homology (H) domains-1), connects to the regulation of cytoskeletal dynamics and focal contact assembly. However, the downstream connections of GRAF and ASA1 remain undefined 4. The C-terminal domain of also encompasses the FAT region, which promotes the colocalization of with integrins at focal contacts (BOX 1).Whereas it was first hypothesized that might bind activation and phosphorylation The best-characterized phosphorylation event is AUTOHOSHORYLATION at Tyr397, which can occur in either cis or trans 35. hosphorylation of at Tyr397 creates a motif that is recognized by various SH2-DOMAINcontaining proteins, such as SRC-FAMILY KINASES (SFKs), phospholipase Cγ (LCγ), suppressor of cytokine signalling (SOCS), growth-factor-receptor-bound protein-7 (GRB7), the Shc adaptor protein, p120 RasGA, and the p85 subunit of phosphatidylinositol 3-kinase (I3K) 4,18,31,33 (FIG. 2).It is not known whether these different signalling proteins differentially bind to Tyr397-phosphorylated in response to particular cell stimuli or whether simultaneously there are different complexes with a larger pool of activated. In this respect, we favour a sequential association model whereby the binding of cellular Src (hereafter referred to as Src) to initiates signalling (discussed below) and the association of SOCS with is a terminal event that leads to ubiquitin-mediated degradation of 36. For integrin-, growth factor- and G-protein-linked stimuli that promote cell motility, it is the transient recruitment of SFKs into a signalling complex with that is one of the first events associated with activation 18.roline-rich tyrosine kinase-2 (YK2) is related to and shares a similar domain structure (FERM, kinase, proline-rich and FAT domains) as well as common phosphorylation sites (BOX 2).The binding of SFKs to YK2 that is phosphorylated at Tyr402 is also associated with YK2 activation. However, and YK2 possess distinct signalling roles in cells, partly owing to differential binding of target proteins to the FERM and FAT domains of and YK2, respectively. Additionally, YK2 is preferentially expressed in cells of the endothelium, central nervous system and haematopoietic lineages; YK2 activation is sensitive to intracellular Ca 2+ signals; and YK2 is only weakly activated in response to the binding of α 5 β 1 -integrin to fibronectin, whereas is strongly activated 37. The difference in α 5 β 1 -mediated activation of versus YK2 is directly related to the FAT-mediated localization of at focal contacts compared with a perinuclear distribution of YK2 in cells 38.Although YK2 and can bind SFKs and can activate common signalling pathways, the differential binding activities of the FERM and FAT domains might limit the functional redundancy of these TKs in cells. The activity of is dependent on integrinmediated cell adhesion. Models of integrin-mediated intermolecular activation are based on the fact that mutants can compete for integrin association and 58 JANUARY 2005 VOLUME 6

4 Ezrin DGF receptor, ETK, EGF receptor FERM Figure 2 Focal adhesion kinase domain structure and phosphorylation sites. Focal adhesion kinase () contains a FERM (protein 4.1, ezrin, radixin and moesin homology) domain, a kinase domain and a focal adhesion targeting (FAT) domain. The FERM domain mediates interactions of with the epidermal growth factor (EGF) receptor, platelet-derived growth factor (DGF) receptor, the ETK tyrosine kinase and ezrin, and the FERM domain can be conjugated to SUMO (small ubiquitin-related modifier) at Lys152. The FAT domain recruits to focal contacts by associating with integrin-associated proteins such as talin and paxillin. It also links to the activation of Rho GTases by binding to guanine nucleotide-exchange factors (GEFs) such as p190 RhoGEF. contains three proline-rich regions (RR1 3), which bind Srchomology-3 (SH3) domain-containing proteins such as p130cas, the GTase regulator associated with (GRAF) and the Arf-GTase-activating protein ASA1. is phosphorylated () on several tyrosine residues, including Tyr397, 407, 576, 577, 861 and 925. Tyrosine phosphorylation on Tyr397 creates a Src-homology-2 (SH2) binding site for Src, phospholipase Cγ (LCγ), suppressor of cytokine signalling (SOCS), growth-factor-receptorbound protein 7 (GRB7), the Shc adaptor protein, p120 RasGA and the p85 subunit of phosphatidylinositol 3-kinase (I3K). hosphorylation of Tyr576 and Tyr577 within the kinase domain is required for maximal catalytic activity, whereas the binding of -family interacting protein of 200 kda (FI200) to the kinase region inhibits catalytic activity. phosphorylation at Tyr925 creates a binding site for GRB2. ADATOR ROTEINS roteins that augment cellular responses by recruiting other proteins to a complex. They usually contain several protein protein interaction domains. G-ROTEIN-COULED RECETOR A seven-helix membranespanning cell-surface receptor that signals through heterotrimeric GT-binding and GT-hydrolysing G-proteins to stimulate or inhibit the activity of a downstream enzyme. SRC-HOMOLOGY (SH)3-DOMAIN A protein sequence of 50 amino acids that recognizes and binds sequences that are rich in proline. GTase-ACTIVATING ROTEIN (GA). A protein that stimulates the intrinsic ability of a GTase to hydrolyse GT to GD. Therefore, GAs negatively regulate GTases by converting them from active (GT-bound) to inactive (GD-bound). Tyr397 Tyr576 Tyr577 Tyr861 Tyr925 Kinase domain FAT Lys152 RR1 FI200 RR2 RR3 SUMO p120 RasGA, GRB7, Shc, LCγ, p85, Src, SOCS p130cas, ASA1, GRAF GRB2, p190 RhoGEF, talin, paxillin can inhibit endogenous activity 4 and that kinaseinactive can become transphosphorylated on Tyr397 in cells 23. Tyr397 phosphorylation promotes Src binding, which leads to the conformational activation of Src and results in a dual-activated Src signalling complex 18.Within this Src complex, Src phosphorylates at Tyr861, and this is associated with an increase in SH3-domain-mediated binding of p130cas to the C-terminal proline-rich regions 39. Activated Src also phosphorylates at Tyr925, which creates an SH2-binding site for the GRB2 adaptor protein. GRB2 binding to is one of several connections that lead to the activation of Ras and the extracellular signal-regulated kinase-2 (ERK2)/mitogen-activated protein kinase (MAK) cascade 18. ERK2 phosphorylation and the subsequent activation of myosin light chain kinase can modulate focal contact dynamics in motile cells 3, as well as generate both proliferative and survival signals inside cells 31. Regulation of catalytic activity Src-mediated transphosphorylation of within the kinase domain ACTIVATION LOO at Tyr576 and Tyr577 promotes maximal catalytic activation 31.Mutation of within this loop produces mutants with either enhanced or refractory activities. One such mutant, super, contains a Lys to Glu substitution at residues 578 and 581, and results in a protein with adhesion-independent activity 40.However, the phosphorylation of downstream targets in superexpressing cells remained adhesion dependent, which thereby reinforced the role of integrins in the regulation of signalling. Results showing that catalytic activity can be modulated by either posttranslational or mutational changes in activation-loop residues are consistent with crystal structure analysis of the AT-bound kinase domain of, which shows a disordered activation-loop conformation 41.As the crystal structure of the kinase domain of also showed the presence of an unusual disulphide bond between Cys456 and Cys459 in a regulatory region, conformational changes or protein-binding interactions might also function to modulate the activation state of. This model is supported by findings that cellular proteins such as -interacting protein of 200 kda (FI200) bind to the kinase domain of and inhibit activity 42.Additionally, evidence is accumulating that intramolecular constraints also have a role in the regulation of activity. There are alternatively spliced isoforms of in which amino-acid additions surrounding the Tyr397 site promote a change in the kinetics of activation (as measured by Tyr397 phosphorylation) from a primarily trans-intermolecular reaction to a cis-intramolecular reaction 35.Although alternative splicing of does not alter the FERM domain residues of, truncation or removal of the FERM domain does result in enhanced catalytic activity 35.As binding of proteins such as ezrin or the GUANINE NUCLEOTIDE-EXCHANGE FACTOR (GEF) TRIO to the FERM domain result in enhanced activity 26,43, and as the FERM domain can bind in trans to the catalytic domain, resulting in the inhibition of activity 44, it is possible that binding interactions or conformational changes in the FERM domain might function to release cis-inhibitory constraints on catalytic activation. The activity of can also be modulated positively 45 or negatively 46 by the action of protein-tyrosine phosphatases (Ts). Studies using Tα-deficient fibroblasts showed that this phosphatase was required for maximal stimulation of Src catalytic activity by β 1 -integrins, and that Tα functioned as an upstream regulator of Tyr397 phosphorylation 45.This result is consistent with the potential intermolecular activation of by Src. As Src can also become activated through direct interaction with the cytoplasmic domains of β-integrins 47, these types of result reinforce the fact that Tyr397 phosphorylation of might not always reflect catalytic activity that is mediated by autophosphorylation. p130cas and paxillin as targets of In addition to promoting maximal activation, the recruitment of Src into a Src signalling complex functions to facilitate the phosphorylation of various -associated proteins, as many targets are also independent binding partners and phosphorylation targets of Src. Two of the best-characterized target proteins of Src-mediated phosphorylation are p130cas and paxillin 31,32,48. SH3-mediated binding of p130cas to is linked to enhanced tyrosine phosphorylation of NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 6 JANUARY

5 Box 2 The -related kinase YK2 roline-rich tyrosine kinase-2 (YK2) shares a similar domain arrangement with focal adhesion kinase () (see figure), with 60% sequence identity in the central kinase domain, conservation of proline-rich regions (RRs), and identical positions of four tyrosine phosphorylation sites. YK2 tyrosines 402, 579, 580 and 881 correspond to tyrosines 397, 576, 577 and 925, respectively. hosphorylation of YK2 Tyr402 and Tyr881 create Src-homology-2 (SH2) binding sites for Src and growth-factor-receptorbound-2 (GRB2), respectively. YK2 contains a C-terminal focal adhesion targeting (FAT) domain that binds to paxillin 53.However, YK2 shows perinuclear distribution and is not strongly localized to focal contacts in many cells 37. The substitution of the C-terminal domain to YK2 facilitated the colocalization of this YK2 chimaera to β 1 -integrin-containing focal contacts 38,which indicates that there are biologically relevant binding differences between and YK2. For instance, the C-terminal domain uniquely binds the integrin-associated protein talin 114, and YK2 but not binds the actin-associated protein gelsolin 115.Although YK2 can be activated by integrins, this is dependent on integrin-mediated activation of Src-family kinases 116,117. The 40% sequence similarity between the N-terminal FERM (protein 4.1, ezrin, radixin and moesin homology) domains of YK2 and also accounts for differential association with target proteins 118.What remains unknown is why YK2 activity is highly dependent on intracellular Ca 2+ levels and how YK2 associates with members of the Janus kinase family 37,119 properties that are not shared by. As YK2 regulates several signalling events that are crucial for macrophage 120 and monocyte morphology 121, and the yk2-null phenotype results in a MARGINAL ZONE B-cell developmental defect 122, there is probably a unique role for YK2 in mediating haematopoietic cell responses to chemokine stimuli. AUTOHOSHORYLATION The transfer of a phosphate group by a protein kinase either to a residue in the same kinase molecule (cis) or to a residue in a different kinase molecule but of the same type (trans). SH2 DOMAIN A protein motif that recognizes and binds tyrosinephosphorylated sequences, and thereby has a key role in relaying cascades of signal transduction. SRC-FAMILY KINASES Kinases that belong to the Src family of tyrosine kinases, the largest of the non-receptortyrosine-kinase families. Src Tyr397 Tyr576 Tyr577 GRB2 Tyr861Tyr925 FERM Kinase domain FAT Sequence similarity RR1 RR2 RR3 <10% ~40% ~60% ~40% YK2 FERM Kinase domain FAT RR1 Tyr402 Tyr579 Tyr580 RR2 RR3 Tyr881 Src GRB2 p130cas at multiple sites, which promotes SH2-mediated binding of the Crk adaptor protein to p130cas. Signalling downstream of p130cas results in increased activity of Rac, enhanced MEMBRANE RUFFLING or lamellipodia formation, and the promotion of cell motility or invasion 17,49,50 (FIG. 3). axillin is phosphorylated by Src on Tyr31 and Tyr118, and this can also promote SH2-mediated binding of Crk to paxillin 48,51. Overexpressing paxillin that is mutated at these phosphorylation sites inhibits the turnover of focal contacts 6 and cell motility 52,which therefore supports the presence of multiple routes for Src-mediated signalling in modulating the dynamics of cell adhesion sites. Regulated targeting of to focal contacts It is the C-terminal FAT domain of that facilitates the linkage to integrins and focal contacts. The FAT domain adopts a four-helix bundle structure that contains binding sites for integrin-associated proteins such as paxillin 53.oint mutations in the FAT domain of that disrupt paxillin binding also prevent the association of with β 1 -integrin and the localization of to focal contacts 38.axillin binding is mediated by two leucine-rich peptide regions in paxillin that are known as LD MOTIFS,which interface with hydrophobic surface grooves on the FAT domain 54,55.Interestingly, the SH2 binding site for GRB2 at Tyr925 partially overlaps with one of the two paxillin LD-motif binding sites in the FAT domain 54, and localization studies of phosphorylated have shown that Tyr925-phosphorylated might be selectively excluded from focal contact sites 56.Overexpression of a Tyr925he mutant of resulted in strong focal contact distribution 56, and in activated Src-expressing cells, Tyr925he blocks the turnover of focal contacts (V. Brunton, personal communication; see note added in proof). As NMR analyses have shown that the FAT domain can undergo conformational rearrangements that might selectively promote either Tyr925 phosphorylation and/or paxillin binding 57, it is possible that Src-mediated phosphorylation of on Tyr925, and subsequent GRB2 binding, could displace paxillin, promote the dissociation of from focal contacts, and subsequently lead to focal contact turnover through undefined mechanisms (FIG. 3). Ser910 within the FAT domain is phosphorylated during mitosis 58 and after growth factor stimulation of cells. Ser910 is phosphorylated by ERK2 and this is also associated with reduced paxillin binding to 59.So, Src-mediated phosphorylation of Tyr925 on and GRB2 binding leading to ERK2 activation, coupled with the feedback of ERK2-mediated Ser910 phosphorylation, could potentiate the release of from focal contacts. Alternatively, Src-mediated phosphorylation of paxillin at Tyr118 promotes the binding of ERK2 to paxillin 60, and ERK2-mediated phosphorylation of paxillin can facilitate binding to paxillin and can enhance activation 60,61.Therefore, we speculate that there might be a regulatory cycle in which Src activation and signalling to ERK2 can function first to promote release from existing focal contacts and then, through ERK2-mediated phosphorylation of paxillin, to promote re-binding and activation at new or different focal contacts in a migrating cell (FIG. 3). Regulation of focal contact dynamics Lessons from / and SH2 / cells. In analyses monitoring the formation of focal contacts, was found to be one of the first signalling proteins to be recruited to these sites 62.Although recruitment to focal contacts is associated with increased tyrosine phosphorylation 63,focal contacts readily form in -null ( / ) fibroblasts, which indicates that activity is not essential for the process of focal-adhesion formation 5.However, focal contacts in / cells form primarily around the cell periphery, enmeshed in a cortical actin ring, and do not undergo a normal maturation cycle 64.In normal fibroblasts, peripheral immature focal contacts become connected to longitudinal stress fibres in cells and undergo actin contractility-mediated maturation during cell polarization 63. re-expression in 60 JANUARY 2005 VOLUME 6

6 Membrane Focal contacts FERM FERM Src Rac SH2 Tyr397 Tyr576 Tyr577 Kinase domain Kinase domain Migration p130cas SH3 -X-X- Release Figure 3 Focal adhesion kinase () Src signals that regulate cell motility and focal contact localization. Integrin clustering promotes autophosphorylation () at Tyr397, which creates a binding site for the Src-homology (SH)2 domain of Src. Src-mediated phosphorylation of at Tyr576 and Tyr577 promotes maximal catalytic activity. Active Src facilitates SH3-mediated binding of p130cas to and its subsequent phosphorylation. Crk binding to phosphorylated p130cas facilitates Rac activation, lamellipodia formation and cell migration. axillin binding to the focal adhesion targeting (FAT) domain is important for focal contact localization. Src-mediated phosphorylation of at Tyr925 creates an SH2 binding site for the growth-factor-receptor-bound protein 2 (GRB2) adaptor protein, which leads to the activation of Ras and the extracellular signal-regulated kinase-2 (ERK2) cascade. The GRB2 and paxillin binding sites within the FAT domain overlap and Tyr925-phosphorylated might be selectively released from focal contacts. ERK2 activation promotes phosphorylation at Ser910, which is also associated with decreased paxillin binding to. Within focal contacts, Src-mediated phosphorylation of paxillin at Tyr118 promotes ERK2 binding. ERK2-mediated phosphorylation of paxillin can facilitate binding to paxillin and enhances activation. So, there might be a cycle whereby Src- and ERK2-mediated phosphorylation of promotes its release from focal contacts and ERK2-mediated phosphorylation of paxillin promotes the association of unphosphorylated with paxillin at new or growing focal contact sites. Crk Tyr925 Ser910 GRB2 Tyr925 FAT FAT Integrins α β Tyr118 ERK2 axillin ERK2 phosphatase SH2 (SH2-domain-containing protein tyrosine phosphatase 2). SH2 / cells have increased activity, but they also show an accumulation of immature focal contacts and similar refractory migration defects to / cells 67.Hyperactive in SH2 / cells results in an increase in the levels of Tyr12-phosphorylated α-actinin, which thereby reduces the crosslinking of stress fibres and prevents the maturation of focal contacts 68.In SH2 / cells, there is a high level of focal contact turnover 68,whereas in / cells, focal contact turnover and maturation are inhibited 8.So,both SH2 / and / cells show an accumulation of immature focal contacts, but through different mechanisms. These studies support the importance of expression and the precise temporal regulation of activity as important factors that control the dynamics of focal contacts. axillin /, p130cas / and Src / cells. In addition to / and SH2 / cells, fibroblasts that contain null mutations for various other focal-contact-associated proteins also show altered dynamics of focal contact maturation, cell spreading defects and refractory cell motility responses (TABLE 1).Time-lapse analyses showed that the incorporation of labelled paxillin into focal contacts of /, paxillin /, p130cas / or SYF / (Src /, Yes / and Fyn / ) cells did not significantly differ compared to normal fibroblasts 6.However, the rate of focal contact disassembly was much slower in these null cells. Analyses of / cells showed that disassembly was dependent on Tyr397 phosphorylation; SYF / cells showed that Src kinase activity was required; and studies with paxillin / cells indicated that the integrity of the Tyr31 and Tyr118 paxillin phosphorylation sites were needed to promote focal contact turnover 6.As the expression of constitutively active Src in / cells can promote focal contact turnover and increased cell motility 17,69, these combined analyses support the conclusion that, in normal cells, integrin- and -mediated control of Src activity is a key event that promotes focal contact dynamics. MARGINAL ZONE A region in the spleen in which white blood cell precursors such as B-cells, granulocytes, macrophages and plasma-cells reside or transit through during primary or secondary immune responses. ACTIVATION LOO A conserved structural motif in kinase domains, which needs to be phosphorylated for full activation of the kinase. GUANINE NUCLEOTIDE- EXCHANGE FACTOR A protein that facilitates the exchange of GD for GT in the nucleotide-binding pocket of a GT-binding protein. / cells promotes the reorganization of the immature focal contacts, which allows for their connection to actin stress fibres, therefore mediating cell contractility and cell polarization 64. Mechanistically, these alterations in focal contacts and actin structures involve the regulation of the activity of α-actinin, a protein that promotes actin crosslinking and that has an important role in maintaining the linkage between focal contacts and stress fibres 1,65 (FIG. 4). phosphorylates α-actinin at Tyr12, which results in reduced α-actinin binding to actin 66. α-actinin is not phosphorylated in / cells, so this signalling linkage to α-actinin might underlie some of the maturation defects, as well as turnover dynamics, of focal contacts in / cells 66.It is possible that the lack of α-actinin phosphorylation might be associated with the presence of focal contacts enmeshed in a cortical actin ring at the / cell periphery. This hypothesis is further supported by studies of cells that lack the tyrosine Src and proteolysis. In addition to signalling events that are associated with the phosphorylation of α-actinin, p130cas or paxillin, Src signalling can affect focal contact dynamics through the regulation of both extracellular and intracellular proteolytic events. Inhibition of activity in human carcinoma cells or Src-transformed cells, or stable re-expression in / cells, can alter the expression and activation of MATRIX METALLOROTEINASES (MMs) 16,17,22.The influence of on MM regulation is associated with signalling from Ras to ERK2 and from Rac to Jun N-terminal kinase (JNK). Activation of MMs at the LEADING EDGE of migrating cells functions to promote matrix proteolysis, which leads to the extracellular release of integrin matrix contacts and thereby facilitates focal contact turnover 70. The intracellular linkage of focal contacts to the actin cytoskeleton is also regulated by calpain-mediated proteolysis 71.Calpain can cleave constituents of focal NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 6 JANUARY

7 Actin stress fibres Actin stress fibres Assembly Cdc42 Tyr256 AR2/3 Tyr256 N-WAS Increased α-actinin crosslinking α-actinin MLCK ROCK Rho α-actininp190 RhoGEF MLC phosphatase Cdc42 Tyr256 N-WAS Reduced α-actinin crosslinking α-actinin Tyr12 GRAF Rho p190 RhoGEF N-WAS Focal contact Nucleus Focal contact MEMBRANE RUFFLE A process that is formed by the movement of lamellipodia that are in the dynamic process of folding back onto the cell body from which they previously extended. LD MOTIF A short sequence found within proteins that has the consensus sequence LDXLLXXL and functions as a protein-binding interface. MATRIX METALLOROTEINASES roteolytic enzymes that degrade the extracellular matrix and have important roles in tissue remodelling and tumour metastasis. LEADING EDGE The thin margin of a lamellipodium that spans the area of the cell from the plasma membrane to a depth of about 1 µm into the lamellipodium. Figure 4 Focal adhesion kinase promotes cytoskeletal fluidity. Stress fibres and cortical actin are continuously destabilized/stabilized by focal adhesion kinase ()-regulated processes. Normally, the actin cytoskeleton exists in a semi-solid state, owing to a high degree of α-actinin-mediated crosslinking of stress fibres, which are tethered and exert tension at focal contacts (left panel). Conversion to a more soluble state (right panel) is promoted by phosphorylation () on Tyr12 of α-actinin, which results in reduced crosslinking and the release of actin stress fibres from focal contacts. Cytoskeletal fluidity is also regulated by the effects of on Rho-family GTases and on the neuronal Wiskott Aldrich syndrome protein (N-WAS). phosphorylates Cdc42-activated N-WAS at Tyr256, thereby retaining phosphorylated N-WAS in the cytoplasm where it can affect AR2/3-mediated actin polymerization. Through associations with Rho GTase-activating proteins (GAs) and Rho guanine nucleotide-exchange factors (GEFs), can regulate actomyosin stress fibre polymerization. Reduced tension can be attributed in part to increased RhoGA activity of GTase regulator associated with (GRAF). Conversely, can promote cytoskeletal tension through phosphorylation and activation of p190 RhoGEF. Subsequent Rho activation indirectly regulates myosin light chain (MLC) phosphorylation through Rho-associated kinase (ROCK) phosphorylation of MLC phosphatase, which leads to increased MLC kinase (MLCK) activity through the downregulation of MLC phosphatase activity. contacts, such as talin and, and calpain-4 / cells have an increased number of peripheral focal contacts 72. Calpain is not appropriately activated in / cells 21, and this defect might be due in part to ERK2 activation being required for calpain function 73.Notably, re-expression in / cells promotes the formation of a complex between calpain, ERK2 and activated Src 21.Restoration of calpain activity in / cells requires specific phosphorylation events: a form of that is mutated at several phosphorylation sites can form a complex with calpain and ERK2, but it does not restore full calpain activity in contrast to cells in which wild-type is re-expressed 74.So, signalling is connected to the increased turnover of focal contacts through calpain activation. As calpain is a Ca 2+ -dependent protease, and activation is associated with local Ca 2+ -flux-induced disassembly of focal contacts 75, the calpain linkage might be selectively activated at either cell protrusions or tail retraction sites in motile cells. effects on GTases and actin The activity of Ras and the Rho-family GTases Rho, Rac and Cdc42 is positively regulated by GEFs and negatively regulated by GAs. As mentioned above, a number of studies have shown that Src-mediated phosphorylation events can lead to the activation of Ras ERK2 and Rac JNK signalling cascades to promote increased cell migration and invasion 18.In a recently discovered mechanism, overexpression facilitated the SH2-mediated binding and sequestering of p120 RasGA, which diminished the association of p120 RasGA with active Ras 76 and thereby led to Ras activation. In / cells, the intrinsic GTase activity of RhoA is elevated 8, and pharmacological inhibitors of Rho-associated kinase (ROCK) a substrate of Rho partially reverse the polarization defects of / cells 77.Integrin signalling can suppress RhoA activity by tyrosine phosphorylation of p190 RhoGA (which increases its GA activity) 78. Likewise, stable 62 JANUARY 2005 VOLUME 6

8 Table 1 henotypes associated with null mutations in focal contact proteins Cell Embryonic Focal contact Focal contact Integrin-stimulated tyrosine Reference phenotypes (lethality) day formation turnover migration phosphorylation / (p53 / ) 8.5 Increased immature Inhibited Inhibited NA 5 SYF / 9.5 No change Inhibited Reduced ptyr397 reduced 123 p130cas / No change Inhibited Reduced No difference 124 axillin / 9.5 Increased size Decreased Inhibited ptyr397 reduced 125 Vinculin / 10.0 Decreased size ND Stimulated Increased activity 126 Tα / None Delayed ND Reduced ptyr397 reduced 45 SH2 / Increased immature Elevated Inhibited Increased activity 67,68 in suspension Calpain-4 / 10.0 Larger Reduced Decreased No change 72 This table summarizes the phenotypes of fibroblasts that are derived from mice that are null for various proteins associated with focal contacts. and paxillin are involved in the formation of focal contacts, whereas, Src-family kinases (Src, Yes and Fyn; SYF), p130cas and calpain are also involved in focal contact turnover. Except for vinculinnull cells, the lack of any of the above proteins results in impaired integrin-stimulated cell migration., focal adhesion kinase; NA, not applicable; ND, not determined; Tα, protein tyrosine phosphatase-α; ptyr, phosphotyrosine; SH2, Src-homology (SH)2-containing phosphotyrosine phosphatase-2. AR2/3 COMLEX A complex that consists of two actin-related proteins AR2 and AR3, along with five smaller proteins. When activated, the AR2/3 complex binds to the side of an existing actin filament and nucleates the assembly of a new actin filament. The resulting branch structure is Y-shaped. GANGLIOSIDE An anionic glycosphingolipid that carries, in addition to other sugar residues, one or more sialic acid residues. LIID RAFTS Lateral aggregates of cholesterol and sphingomyelin that are thought to occur in the plasma membrane. re-expression in / cells decreased RhoA activity 8 and enhanced p190 RhoGA tyrosine phosphorylation 17.In other cell types, activation and tyrosine phosphorylation are associated with RhoA activation and the formation of stress fibres 18.This connection could be mediated by binding to, and phosphorylating, p190 RhoGEF 34.In neuronal development, signalling through p190 RhoGEF controls axonal branching and synapse formation 79.Although -mediated activation of p190 RhoGEF is a direct route to RhoA activation, the formation of distinct signalling complexes will probably influence whether activation leads to increased or decreased RhoA activity in cells (FIG. 4). In addition to affecting the activity of Ras, Rac and Rho, can influence the function of Cdc42 through binding and phosphorylation of the Cdc42 effector Wiskott Aldrich syndrome protein N-WAS (neuronal WAS) 80.N-WAS, which, in contrast to its name, is ubiquitously expressed, regulates the actin cytoskeleton through activation of the AR2/3 COMLEX 3.Interestingly, only associates with Cdc42-activated N-WAS, and does not itself activate N-WAS. Although phosphorylation of N-WAS at Tyr256 does not affect N-WAS activity towards AR2/3, it does seem important for maintaining a cytoplasmic distribution of N-WAS and for promoting cell motility 80.As Cdc42 regulates actin dynamics in cellular projections, the interaction of with Cdc42-activated N-WAS might couple actin polymerization with membrane protrusion during cell motility (FIG. 4). and microtubules Integrating factors coordinate the regulation of microtubule structures and the actin cytoskeleton during cell motility. Microtubules are important in the establishment and maintenance of cell polarity, and the Rho effector Diaphanous (mdia) functions to stabilize microtubules at the leading edge of migrating cells 81.Integrin-mediated activation of is required for microtubule stabilization by the Rho mdia signalling pathway 7 (FIG. 5).This is partly the result of the -regulated localization of a lipid-raft marker, GANGLIOSIDE GM1, to the leading edge of motile cells. It is hypothesized that the lipid environment at the leading edge preferentially localizes microtubule capping or bridging proteins, which stabilize the association of microtubules with cortical receptors 82. This regulation of a distinct membrane lipid environment by or integrin signalling 83 also functions to promote Rac signalling by maintaining a suitable lipid environment that facilitates the interaction of Rac and effectors such as p21-activated kinase (AK) 84. Interestingly, -stimulated phosphorylation of Ser298 in MAK/ERK kinase-1 (MEK1, also known as MAK kinase-1) by AK is a secondary route that leads to ERK2/MAK activation 85. In neuronal cells, a fraction of colocalizes with a distinct microtubule structure that arises from microtubule-organizing centres and that extends around the nucleus in a branched fork-like form termed a microtubule fork 86.Microtubule forks are believed to promote nuclear re-positioning in the direction of cell movement. Cyclin-dependent kinase-5 (CDK5) phosphorylates at Ser732 in post-mitotic neurons, and antibodies that recognize Ser732-phosphorylated specifically stain microtubule fork structures near the nucleus 86.Neurons that are devoid of CDK5 or that express a mutant in which Ser732 cannot be phosphorylated show a malformed microtubule fork, impaired nuclear movement and altered neuronal development positioning in vivo 86.Whereas the molecular mechanisms that link Ser732 phosphorylation to the localization and organization of microtubule fork structures remain to be defined, this observation is the first of its kind and supports studies that link phosphorylation to enhanced neuronal cell migration 87. and membrane composition The polarization of migrating cells requires membrane modification as well as changes in the underlying cytoskeleton. In addition to the role of in the translocation of LIID RAFT components 7, Src signalling is involved in the modification of phosphatidylinositol lipids, and differentially phosphorylated lipid NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 6 JANUARY

9 ADHERENS JUNCTION A cell cell adhesion complex that contains classical cadherins and catenins that are attached to cytoplasmic actin filaments. TIGHT JUNCTION A circumferential ring at the apex of epithelial cells that seals adjacent cells to one another. Tight junctions regulate solute and ion flux between adjacent epithelial cells. tdins tdins(4) tdins(4,5) 2 IKIγ I3K Talin p85 Vinculin axillin Figure 5 Focal adhesion kinase influences phospholipid and microtubule structures. The phospholipid kinases that have a role in the modification of phosphatidylinositol (tdins) cooperate with focal adhesion kinase () at several levels. Type I tdins phosphate kinase-γ (IKIγ) associates with and talin, and promotes the conversion of tdins-4-phosphate (tdins(4)) to tdins-4,5-bisphosphate (tdins(4,5) 2 ). IKIγ is phosphorylated by, which leads to increased IKIγ activity and increased generation of tdins(4,5) 2. The binding of tdins(4,5) 2 to talin and vinculin is associated with the formation of focal contacts. tdins(4,5) 2 can be converted to tdins-3,4,5-trisphosphate (tdins(3,4,5) 3 ) by tdins 3-kinase (I3K). The regulatory p85 subunit of I3K binds to at Tyr397, which leads to I3K activation by 112. Directional motility requires the generation of phospholipid components such as tdins(4,5) 2 and tdins(3,4,5) 3. Integrin and signalling also promote the translocation of specific components of lipid rafts to membranes. The stabilization of lipid rafts through integrin signalling facilitates the coupling of Rac to target proteins. -mediated translocation of the lipid ganglioside GM1 to the membrane, which is mediated through the activation of the Rho GTase effector Diaphanous (mdia), regulates microtubule polarity at the leading edge of motile cells. Microtubule polarization and Rac activation contribute to the formation of membrane ruffles and stable lamellipodia. tdins(3,4,5) 3 New membranes Membrane component mdia GM1 hospholipid modification by phosphorylation Formation of membrane proximal contacts Microtubule stabilization Lipid rafts intermediates function as binding sites for signalling proteins that are involved in the formation of focal contacts (FIG. 5). hosphatidylinositol-4,5-bisphosphate (tdins(4,5) 2 ) binds to and controls the assembly of proteins such as α-actinin, vinculin and talin into focal contacts 2.As the binding of the talin FERM domain to β-integrin cytoplasmic tails is enhanced by tdins(4,5) 2 (REF. 88), and the talin rod domain binds vinculin and actin 89,a link between integrins, focal contact formation and the actin cytoskeleton is established. The type I phosphatidylinositol phosphate kinase-γ (IKIγ) is an enzyme that makes tdins(4,5) 2 and it is targeted to focal contacts by an association with the talin FERM domain 90.IKIγ is Rac Rac Integrins Stable lamellipodia Increased membrane fluidity α β phosphorylated by a Src complex, which facilitates increased IKIγ activity (and, therefore, increased production of tdins(4,5) 2 ) and increased IKIγ association with talin 91.In this manner, signalling is connected to the formation of focal contacts and the spatial regulation of tdins(4,5) 2 generation. However, the integrin and IKIγ binding sites within the talin FERM domain overlap, which implies that IKIγ binding might displace talin from integrin tails 92.To this end, -enhanced Src-mediated phosphorylation of IKIγ on Tyr644 creates a high affinity binding site for the talin FERM domain, which displaces β-integrin binding from talin FERM 93.So, although Src activity could promote the production of tdins(4,5) 2 and the formation of focal contacts by enhancing the activity of IKIγ, subsequent phosphorylation of IKIγ by activated Src might break the talin integrin linkage and promote the turnover of focal contacts. and intercellular contacts Another biological context in which signalling has been associated with the formation or turnover of contacts is cadherin-based cell cell junctions. Cadherins are transmembrane proteins that mediate Ca 2+ -dependent homophilic protein protein attachments between cells and that are also linked to the actin cytoskeleton through interaction with α- or β- catenins 94.Downregulation of E-cadherin-based ADHERENS JUNCTIONS is a hallmark of malignant and invasive carcinomas, and the activity of the Src complex promotes the disruption of colon carcinoma cell homotypic adhesions 95.Importantly, expression of a protein that is mutated at five tyrosine phosphorylation sites (Tyr407, 576, 577, 861 and 925) blocked the Src-mediated disruption of colon carcinoma E-cadherin-based contacts, thereby implying that phosphorylation-dependent signalling through was required 96.In an opposite manner, overexpression of a kinase-defective mutant of blocked the accumulation of peripheral E-cadherin in endothelial cells that were subjected to a hyperosmolar challenge (a stimulus that promotes increased E-cadherin-based TIGHT- JUNCTION barrier formation) 97.These results imply that signalling has a role in both the formation and turnover of E-cadherin-based contacts. As opposed to E-cadherin function, N-cadherin expression in carcinoma cells is generally associated with a scattered morphology and a migratory or invasive phenotype. Antisense and DOMINANT-NEGATIVE inhibition of showed that expression and activity were needed for the formation of N-cadherin-based cell cell contacts in HeLa cells 19.However,in contradiction, the above study also found that cells with less expression and reduced N-cadherin-mediated cell cell contacts exhibited increased motility when plated as individual cells on a collagen matrix. Whereas much remains to be determined about the molecular role of in either the dissolution or formation of cadherinbased contacts, it is intriguing that the findings so far are somewhat similar to the bi-functional role of in focal contact dynamics. 64 JANUARY 2005 VOLUME 6