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Minireview 647

Molecular mechanisms for assembly through regulation of –protein interactions Andrew P Gilmore and Keith Burridge

Focal adhesions provide a useful model for studying these inhibition experiments it was concluded that tyro- /extracellular matrix interactions and the sine phosphorylation of FA stimulated FA forma- subsequent cytoskeletal reorganization. Recent tion, and that FAK was the important kinase. However, advances have suggested potential mechanisms by recent evidence suggests that FAK does not regulate FA which cells may regulate focal adhesion assembly formation. Cells cultured from FAK-deficient mice are following -mediated cell adhesion. able to form FAs [6], and mouse aortic smooth muscle cells can form FAs without FAK becoming activated [7]. Address: Department of Cell Biology and Anatomy, University of North In addition, displacement of endogenous FAK from FAs Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. of adherent cells, by microinjecting the cells with a domi- Structure 15 June 1996, 4:647–651 nant-negative form of FAK, does not inhibit FA formation (APG and LH Romer, unpublished data). Furthermore, in © Current Biology Ltd ISSN 0969-2126 normal cells, the most prominent FA proteins, including , , integrin and ␣-, are not significantly Focal adhesions (FAs) are discrete regions of a cell in phosphorylated on tyrosine residues. culture where it is most tightly associated with the under- lying extracellular matrix (ECM) [1]. They have become a Unlike FAK, the small GTP binding-protein Rho has been popular model for studying integrin-mediated adhesion. demonstrated to be essential for FA formation [5]. Swiss FAs provide sites of mechanical attachment to the ECM, 3T3 fibroblasts which have been deprived of serum lack and are points at which adhesion-associated signal trans- FAs and stress fibres. Stimulation with neuropeptides such duction is initiated. The latter property has important as bombesin or vasopressin, or with the serum component implications for anchorage-dependent behaviours such as lysophosphatidic acid (LPA), causes these structures to cell proliferation and inhibition of apoptosis. Two recent assemble. Inactivation of Rho by the bacterial toxin C3 exo- reviews have discussed several aspects of FA structure and transferase blocks neuropeptide- and LPA-induced FA for- function [2,3]. Rather than repeating information from mation [5]. Possible downstream effectors of Rho include these reviews, we will discuss potential mechanisms for phosphatidylinositol-5-kinase (PtdIns-5-kinase). Rho stim- FA formation in terms of the regulation of the association ulates PtdIns-5-kinase, which in turn converts phos- of their component proteins. phatidylinositol-4-phosphate (PtdInsP) to phosphatidyl- inositol-4,5-bisphosphate (PtdInsP2) [8]. PtdInsP2 levels in Attachment to the ECM at FAs is mediated by members cells increase upon integrin-mediated cell adhesion and of the integrin family of transmembrane proteins. The when Rho is activated. A number of -binding proteins cytoplasmic components of FAs include cytoskeletal pro- are regulated by PtdInsP2 in vitro, suggesting a potential teins, such as ␣-actinin, vinculin and talin, and signalling mechanism for Rho’s effects on the [9–11]. proteins such as the tyrosine kinase FAK (focal adhesion Two other members of the Rho family, Rac and cdc42, also kinase). Possible interactions between the major compo- regulate assembly of the actin cytoskeleton. Rac stimulates nents of FAs are summarized in Figure 1. Formation of membrane ruffling (lamellipodia), whereas cdc42 induces FAs must involve the coordination of several different the formation of filopodia (microspikes) [12]. The functions events, including actin polymerization, actin stress fibre of these three closely related members of the Ras super- attachment to the membrane at sites of cell–ECM adhe- family are interlinked: cdc42 stimulates Rac, and Rac stim- sion, and stress fibre contraction. Recent evidence, dis- ulates Rho. The mechanisms by which cdc42 and Rac cussed in detail below, has suggested that these processes modulate actin polymerization are not clearly understood, are coordinated in response to stimulation. The different but may involve pathways similar to those mediating the steps involved in FA formation are summarized in effects of Rho. Both the induction of membrane ruffling by Figure 2, and we will discuss each step in detail below. Rac and FA formation by Rho involve co-localization of vin- culin, talin and actin. PtdInsP2-binding peptides derived A number of signal transduction pathways may regulate from the sequence of inhibit Rac-mediated actin FA formation, in particular those pathways involving polymerization in platelets, suggesting that Rac’s effects tyrosine kinases [4] and the GTP-binding protein Rho may also involve PtdIns-5-kinase [13]. [5]. Integrin-mediated cell adhesion induces tyrosine phosphorylation of a number of FA proteins, including Rac and cdc42 activate kinase cascades similar to the FAK. Tyrosine kinase inhibitors, such as herbimycin and Ras/Raf/mitogen activated protein kinase pathway [14]. tyrphostins, inhibit FA and stress fibre formation. From Evidence is accumulating to suggest that Rho also acts 648 Structure 1996, Vol 4 No 6

Figure 1 Figure 2

SIGNALLING PATHWAYS

FIBRONECTIN MATRIX

F-Actin Tensin Paxillin

α-Actinin CRP FAK

Vinculin Zyxin G-Actin

Talin Profilin Fibronectin

Integrin VASP

A model showing how focal adhesion (FA) components may link actin stress fibres to sites of cell–ECM adhesion. The potential interactions between the many components of FAs have been characterized mostly in vitro. The illustration has been simplified to include those proteins which are most likely to be involved in the formation and structural integrity of FAs, and does not include the many signalling proteins regulating downstream effects following cell adhesion. It is clear that a considerable redundancy exists with regard to the association of actin filaments with the cytoplasmic domains of . The fibronectin matrix is extracellular. For further discussion on interactions not dealt with in this review, see [1–3].

A potential model for focal adhesion formation. Focal adhesion through a pathway involving multiple kinases. Tyrphostin formation requires three distinct events which may be regulated inhibits the effect of LPA in stimulating Rho-dependent through the same pathways, such as those involving the GTP-binding stress fibre and FA formation [15]. However, tyrphostin protein Rho. These events may occur simultaneously, but are shown here as independent stages for the sake of clarity. (a) In a suspended has no effect when cells are microinjected with an acti- cell, integrins are not bound to ECM ligand and are dispersed over the vated form of Rho (Rhoval14), suggesting that LPA acts on cell surface. Integrins are not associated with cytoskeletal components (b) a tyrosine kinase upstream of Rho. Rhoval14 stimulation of within the cell, and actin is not polymerized. Following ECM FA formation is inhibited by another kinase inhibitor, binding, actin polymerization extends from sites of cell–ECM adhesion. This may occur through the association of zyxin, VASP and profilin at genistein [16]. Kinases are therefore required for LPA these regions, possibly through interactions with ␣-actinin and integrin. to activate Rho, and for subsequent Rho-mediated FA (c) Integrins associate with cytoskeletal proteins, including talin and formation. A serine/threonine kinase, protein kinase N vinculin, which may stabilize F-actin attachment to the membrane. (PKN), has recently been shown to interact with Rho, and (d) Stimulation of stress fibre contraction causes the integrins to to become phosphorylated in cells following LPA stimula- cluster at the membrane, forming the focal adhesion. tion [17]. This phosphorylation is prevented by treating cells with C3 exotransferase. this high concentration of unpolymerized actin by seques- tering G-actin monomers such that the available pool for Actin polymerization polymerization remains below the critical concentration, Cells contain G-actin at concentrations of around and by capping actin nuclei to block further polymeriza- 50–200 ␮M, whereas the critical concentration for polymer- tion. Cells contain a wide variety of proteins that are ization is around 0.2 ␮M for ATP⋅actin. Cells can maintain capable of carrying out these functions, and the modulation Minireview Focal adhesion assembly Gilmore and Burridge 649

of their activity may be an important part of FA formation. talin, ␣-actinin and F-actin, but only if the antibody used PtdInsP2 regulates some of the proteins that control actin was adhesion inhibitory [21]. Non-inhibitory antibodies assembly, including gelsolin, ␣-actinin and profilin [9–11]. induced co-localization of FAK and tensin, but not the more prominent FA components. A hypothesis to explain Gelsolin has three prominent actions on actin: barbed end these findings is that inhibitory antibodies bind integrin capping, nucleation of polymerization, and filament sever- close to the ligand-binding site and mimic ECM binding. ing. Gelsolin, therefore, can produce a large number of Integrins clustered with non-inhibitory antibodies in the short, capped actin filaments. PtdInsP2 inhibits gelsolin’s presence of soluble ECM ligands also induce co-localiza- interactions with actin, which would make these actin tion of vinculin, talin, ␣-actinin and F-actin. This is one nuclei available for polymerization [10]. ␣-Actinin cross- example of ‘outside-in’ signalling (the regulation of inte- links actin filaments and this action is greatly enhanced by grins’ association with cytoplasmic proteins through their PtdInsP2 [11]. The combined effects of PtdInsP2 on the interactions with extracellular factors). It has been proposed interactions of gelsolin and ␣-actinin with actin would lead that the integrin ␣ and ␤ cytoplasmic domains may interact, to a net increase in F-actin bundle formation. and that activation involves their dissociation (for a detailed discussion, see [2]). So called ‘inside-out’ signalling (the Actin polymerization in cells is clearly a localized phenome- regulation of integrins’ ability to bind ECM ligands by non. Polymerization occurs at the leading edges of cells, factors inside the cell) may involve Rho. Inactivation of and at nascent FAs. A candidate protein for restricting actin Rho inhibits the ability of a number of integrins to promote polymerization to specific sites is VASP (vasodilator-stimu- adhesion and spreading [22]. Integrin-mediated FA forma- lated phosphoprotein). In platelets, VASP is phosphory- tion may require both internal and external factors. lated by cyclic AMP and cyclic GMP dependent protein kinases; this event correlates with the inhibition of platelet The integrin ␤ subunit cytoplasmic domain is sufficient aggregation. VASP localizes along actin stress fibres, and is for integrin FA localization and is the critical part of the particularly concentrated at their ends in FAs. VASP has integrin for the cytoskeleton–membrane linkage [2]. Dif- recently been shown to bind the 83 kDa mammalian homo- ferent regions of the integrin ␤1 cytoplasmic domain inter- logue of zyxin [18], a chicken protein concentrated in FAs act in vitro with a number of FA proteins, including talin, and along stress fibres. It binds to the actin cross-linking ␣-actinin and FAK (see [2] for details). Experiments per- and FA protein ␣-actinin. VASP contains a -rich formed in vivo [23,24] showed that the extreme C termi- domain which binds the G-actin-binding protein profilin nus of the ␤1 cytoplasmic domain appears to be critical for [19]. Profilin may be a major player in promoting favourable both binding to the cytoskeleton and for signal transduc- conditions for actin polymerization, with VASP and zyxin tion at FAs. ␤1 integrins lacking the last 13 amino acids of providing a binding site for profilin at sites of cell adhesion. their C terminus neither localize to FAs, nor support adhe- sion-related signalling, such as FAK phosphorylation [23]. Profilin promotes actin polymerization in a number of If monoclonal antibodies are used to cluster ␤1 integrins ways [20]. These include promoting nucleotide exchange lacking the last 13 amino acids of the C terminus on the (from ADP to ATP) on actin monomers, lowering the criti- cell surface, talin, vinculin or F-actin do not co-cluster, cal concentration of actin required for polymerization, and suggesting that these truncated integrins cannot mediate by desequestering G-actin from thymosin ␤4. In the pres- FA formation [24]. ence of thymosin ␤4, profilin significantly increases actin polymerization in vitro. Profilin association with actin is The binding of integrin to the ECM is not alone sufficient regulated by PtdInsP2, and this in itself is quite complex. for FA formation. Serum-starved Swiss 3T3 cells, in which Profilin–PtdInsP2 cannot bind actin and inhibits PtdInsP2 Rho is inactive, do not have FAs even though they adhere hydrolysis by ␥ (PLC␥). This inhibition to the ECM [5]. FA proteins may undergo conformational can be overcome by tyrosine phosphorylation of PLC␥, changes mediated through Rho-dependent pathways which suggesting that a number of signalling pathways may modulate their affinities for each other. Vinculin binds a overlap at this point to regulate the availability of both number of other FA proteins [3]. Its N-terminal head profilin and PtdInsP2. domain binds talin and ␣-actinin, and its C-terminal tail binds actin, paxillin and acidic . It can also Integrins and actin membrane attachment associate with tensin, although the binding site has not Integrins have received considerable attention with regard been identified. An intramolecular association between its to how their association with the ECM may be regulated C-terminal tail and N-terminal head regulates vinculin’s (for a recent review, see [2]). Recent work has suggested interaction with other proteins [25]. Intact, native vinculin that ECM binding to the extracellular portion of integrins binds talin and actin poorly because the head–tail inter- promotes association of the cytoplasmic domains with action masks the binding sites for these proteins. In vivo the cytoskeleton. Antibody clustering of integrins on the unfolding of vinculin could promote FA formation by surface of cells resulted in co-clustering of vinculin, exposing these binding sites. Vinculin phosphorylation does 650 Structure 1996, Vol 4 No 6

not occur at a stoichiometry sufficient to account for this. contractility in smooth muscle. Lysophosphatidic acid and However, in vitro vinculin binds PtdInsP2, which disrupts thrombin also induce appreciable contraction in fibroblasts vinculin’s head and tail association, thus exposing the talin- [31,32], and stimulate neurite retraction [33]. These effects and actin-binding sites [26]. may be directly mediated by Rho activation [34]. LPA and thrombin both induce a rapid phosphorylation of the Recent work indicates that other FA proteins may be reg- light chains that precedes the onset of contraction ulated in similar ways. The band 4.1 family of proteins, [30], agents which inhibit kinase including talin, ezrin and radixin, are thought to have a (MLCK) disrupt stress fibres and FAs [32,35]. Together common role in linking the actin cytoskeleton to the these results suggest that one of Rho’s actions is to membrane in structures such as FAs and microvilli. These stimulate myosin light chain phosphorylation and thereby proteins share a common N-terminal domain and typically to stimulate contractility. Coupled with the signals that have a highly charged C terminus. Recent work on ezrin, promote actin polymerization and the attachment of F-actin a major protein component of microvilli in epithelial cells, to sites of adhesion on the ECM, this increased contractility suggests that an interaction between its N-terminal and may contribute to the formation of FAs and stress fibres. C-terminal domains may also regulate its role in mediating actin reorganization [27]. This N-terminal–C-terminal Many of the interactions discussed above have been interaction can promote ezrin oligomerization in vivo. dissected in vitro. Now, however, we may be able to start Talin and radixin, found in FAs, may be similarly regu- linking signal transduction pathways which induce FA lated. Talin can form dimers through self association formation with the protein–protein interactions that drive in vitro, and electron microscopic images indicate that their assembly. monomers can either fold up into a globular conformation, or appear elongated and flexible [28]. It is yet to be deter- Acknowledgements We thank Drs Lew Romer and Mike Kinch for comments on the manuscript. mined whether talin can be regulated by such conforma- Due to space limitations, we have been unable to cite much work which is tional changes. The C-terminal domain of talin contains relevant to this area, and we apologize to those researchers whose work multiple binding sites for vinculin, actin and the cytoplas- has not been credited. The authors were supported by a European Molecu- lar Biology Organization postdoctoral fellowship (APG), and by grants from mic domain of integrin [3]. The possible unmasking of the National Institutes of Health (KB). any or all of these sites could clearly promote FA forma- tion in a similar way to that envisaged for vinculin. References 1. Burridge, K., Fath, K., Kelly, T., Nuckolls, G. & Turner, C. (1988). Focal adhesions: transmembrane junctions between the extracellular matrix Stress fibre contractility and the cytoskeleton. Annu. Rev. Cell Biol. 4, 487–525. Interactions between integrins and the ECM do not alone 2. Schwartz, M.A., Schaller, M.D. & Ginsberg, M.H. (1995). Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. cause integrin clustering. Integrins with cytoplasmic 11, 549–599. domain mutations that perturb their association with the 3. Jockusch, B.M., et al., & Winkler, J. (1995). The molecular architecture of focal adhesions. Annu. Rev. Cell Dev. Biol. 11, 379–416. cytoskeleton do not localize to FAs. Agents which disrupt 4. Burridge, K., Turner, C.E. & Romer, L.H. (1992). Tyrosine phosphorylation the actin cytoskeleton, such as cytochalasin D, disrupt of paxillin and pp125FAK accompanies cell adhesion to extracellular FAs and inhibit adhesion-mediated signalling without pre- matrix: a role in cytoskeletal assembly. J. Cell Biol. 119, 893–903. 5. Ridley, A.J. & Hall, A. (1992). The small GTP binding protein rho venting integrin–ECM interactions. In addition, serum- regulates the assembly of focal adhesions and stress fibers in starved Swiss 3T3 cells in which Rho is inactive are response to growth factors. Cell 70, 389–399. adherent but do not possess stress fibres or FAs. 6. Ilic, D., et al., & Aizawa, S. (1995). Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377, 539–544. It was proposed many years ago that stress fibres were con- 7. Wilson, L., Carrier, M.J. & Kellie, S. (1995). pp125FAK tyrosine kinase activity is not required for the assembly of F-actin stress fibres and tractile and could exert tension upon the substrate [29]. focal adhesions in cultured mouse aortic smooth muscle cells. Fibroblasts grown on silicone rubber formed wrinkles in J. Cell Sci. 108, 2381–2391. the substrate due to this tension. Isometric tension gener- 8. Chong, L.D., Traynor-Kaplan, A., Bokoch, G.M. & Schwartz, M.A. (1994). The small GTP-binding protein rho regulates a ated by contraction on a rigid substrate may be the force phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. clustering actin filaments into stress fibres in cultured cells, Cell 79, 507–513. 9. Lassing, I. & Lindberg, U. (1985). Specific interaction between resulting in FA formation at their ends. In support of this phosphatidylinositol 4,5-bisphosphate and profilactin. Nature 314, idea, cells grown in collagen gels will contract the gel over 472–474. several days, and these cells do not possess stress fibres 10. Janmey, P.A. & Stossel, T.P. (1987). Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature 325, 362–364. [30]. Anchoring the gel so that it cannot contract induces 11. Fukami, K., et al., & Takenawa, T. (1992). Requirement of phosphatidyl- isometric tension and the formation of stress fibres by the inositol 4,5-bisphosphate for ␣-actinin function. Nature 359, 150–152. cells, a situation similar to growing cells on a rigid substrate. 12. Nobes, C.D. & Hall, A. (1995). Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53–62. Many of the same agents that induce stress fibre and FA 13. Hartwig, J.H., et al., & Stossel, T.P. (1995). Thrombin receptor ligation and activated rho uncap actin filament barbed ends through formation by activating Rho (lysophosphatidic acid, throm- phosphoinositide synthesis in permeabilized human fibroblasts. bin, vasopressin, endothelin and bombesin) also cause Cell 82, 643–653. Minireview Focal adhesion assembly Gilmore and Burridge 651

14. Coso, O.A., et al., & Gutkind, J.S. (1995). The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signalling pathways. Cell 81, 1137–1146. 15. Nobes, C.D., Hawkins, P., Stephens, L. & Hall, A. (1995). Activation of the small GTP-binding proteins rho and rac by growth factor receptors. J. Cell Sci. 108, 225–233. 16. Ridley, A.J. & Hall, A. (1994). Signal transduction pathways regulating rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J. 13, 2600–2610. 17. Amano, M., et al., & Kaibuchi, K. (1996). Identification of a putative target for rho as the serine-threonine kinase protein kinase N. Science 271, 648–650. 18. Reinhard, M., Jouvenal, K., Tripier, D. & Walter, U. (1995). Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and protein VASP (vasodilator-stimulated phosphoprotein). Proc. Natl. Acad. Sci. USA 92, 7956–7960. 19. Reinhard, M., et al., & Walter, U. (1995). The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins. EMBO J. 14, 1583–1589. 20. Theriot, J.A. & Mitchison, T.J. (1993). The three faces of profilin. Cell 75, 835–838. 21. Miyamoto, S., Akiyama, S.K. & Yamada, K.M. (1995). Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267, 883–885. 22. Laudanna, C., Campbell, J.J. & Butcher, E.C. (1996). Role of rho in chemoattractant-activated leukocyte adhesion through integrins. Science 271, 981–983. 23. Chen, H.-C., Appeddu, P.A., Parsons, J.T., Hildebrand, J.D., Schaller, M.D. & Guan, J.-L. (1995). Interaction of focal adhesion kinase with cytoskeletal protein talin. J. Biol. Chem. 270, 16995–16999. 24. Lewis, J.M. & Schwartz, M.A. (1995). Mapping in vivo associations of cytoplasmic proteins with integrin ␤1 cytoplasmic domain mutants. Mol. Biol. Cell 6, 151–160. 25. Johnson, R.P. & Craig, S.W. (1994). An intramolecular association between the head and tail domains of vinculin modulates talin binding. J. Biol. Chem. 269, 12611–12619. 26. Gilmore, A.P. & Burridge, K. (1996). Regulation of vinculin binding to talin and actin by phosphatidylinositol-4-5-bisphosphate. Nature, in press. 27. Gary, R. & Bretscher, A. (1995). Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell 6, 1061–1075. 28. Molony, L., McCaslin, D., Abernethy, J., Paschal, B. & Burridge, K. (1987). Properties of talin from chicken gizzard smooth muscle. J. Biol. Chem. 262, 7790–7795. 29. Burridge, K. (1981). Are stress fibres contractile? Nature 294, 691–692. 30. Grinnell, F. (1994). Fibroblasts, myofibroblasts and wound contraction. J. Cell Biol. 124, 410–404. 31. Kolodney, M.S. & Elson, E.L. (1993). Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts. J. Biol. Chem. 268, 23850–23855. 32. Chrzanowska-Wodnicka, M. & Burridge, K. (1996). Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J. Cell. Biol., in press. 33. Moolenaar, W.H. (1994). LPA: a novel lipid mediator with diverse biological actions. Trends Cell Biol. 4, 213–219. 34. Jalink, K., van Corven, E.J., Hengeveld, T., Morii, N., Narumiya, S. & Moolenaar, W.H. (1994). Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein rho. J. Cell Biol. 126, 801–810. 35. Volberg, T., Geiger, B., Citi, S. & Bershadsky, A.D. (1994). Effect of protein kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system. Cell Motil. Cytoskeleton 29, 321–338.