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Signal transduction and actin filament organization
66
Signal transduction and actin filament organization Sally H Zigmond Small GTP-binding proteins of the Rho family appear to integrate extracellular signals from diverse receptor types and initiate rearrangements of F-actin. Active members of the Rho family, Rho and Rac, are now joined by Cdc42 which induces filopodia. Downstream of the Rho family proteins, actin polymerization may be induced by an increase in the availability of actin filament barbed ends. Actin organization may be affected by exposure of actin-binding sites on proteins such as vinculin and ezrin.
Address Biology Department, Universityof Pennsylvania, Philadelphia, PA 19104-6018, USA; e-mail: [email protected] Current Opinion in Cell Biology 1996, 8:66-73 © Current Biology Ltd ISSN 0955-0674 Abbreviations EGF epidermal growth factor GTPase-activating protein GAP GEF guanine nucleotide exchange factor LPA lysophosphatidic acid PAK protein-activated kinase PDGF platelet-derived growth factor PI phosphatidylinositol Pl3K phosphatidylinositol 3-kinase phosphatidylinositol 4,5-bisphosphate PIP2 PMA phorbol myristic acetate VASP vasodilator-stimulated phosphoprotein
by the dominant-negative (i.e. GDP-bound form) of the appropriate Rho family member. Thus, injection of dominant-negative Cdc42 blocks the formation of filopodia induced by bradykinin [3°°,4"°], and dominant-negative Rac blocks the formation of ruffles induced by epidermal growth factor (EGF), phorbol myristic acetate (PMA), or bombesin [2",5°]. Inactivation of Rho with the C 3 transferase from Clostridium botulinum blocks the formation of stress fibers induced by lysophosphatidic acid (LPA) [1,6",7]. T h e data suggest that Rho family members mediate these morphological changes that are induced by extracellular agonists. In some cases, the extracellular agonist has also been shown to alter the subcellular location of a Rho family member and/or to stimulate G T P exchange for GDP. However, these changes are usually small [8°,9]. In addition to inducing these specific changes, all members of the Rho family share some functions, and there is cross-talk between family members. Thus, each member of the Rho family can can induce attachment plaques, which are defined by the presence of paxillin and vinculin, two cytoskeletal proteins [3°°]. A given agonist can activate more than one member of the Rho family, and one member of the family can activate another. Thus, Cdc42 can activate Rac, and Rac can activate Rho (see Fig. 1). T h e latter pathway requires the activity of phospholipase A2 and lipoxygenase, presumably to form a leukotriene intermediate [10,11].
Introduction
In response to environmental signals, a cell changes both its shape and its degree of attachment to the substratum. These changes are caused, at least in part, by rearrangements of the actin cytoskeleton. If assembled in long bundles, F-actin supports finger-like protrusions of the plasma membrane known as filopodia; if assembled as a meshwork, it supports sheet-like protrusions known as ruffles or lamellipodia; if present in bundles coupled to attachment plaques, actin 'stress fibers' exert force against the substratum. T h e signals that trigger these different cytoskeletal patterns act through diverse receptors, but it now appears that the pathways leading from these receptors converge on one or more members of the Rho family of small GTP-binding proteins. These proteins are Cdc42, Rac and Rho [1,2"°].
Upstream of the Rho family, only a few elements have been defined. Downstream pathways activate many kinases and transcription factors, and promote cell growth, but it is not known which, if any, of these activities affects the cytoskeleton. However, some pathway eventually stimulates actin polymerization at the barbed (high affinity) end of actin filaments, and some pathway also modulates the association of actin-binding proteins with actin [1,2°°].
Remarkably, when injected in a constitutively active (i.e. a GTP-bound) form into Swiss 3T3 fibroblasts, each of these Rho family members induces unique morphological changes that involve rearrangement of F-actin. Constitutively active Cdc42 induces filopodia [3°'], Rac induces membrane ruffles, and Rho induces stress fibers. When these same morphological changes are induced by extracellular agonists, they can be blocked
Pathways leading to the cytoskeletal changes outlined above are initiated by agonists that bind to a variety of receptors. Bradykinin, bombesin and LPA each bind to a serpentine-type receptor that is linked to a heterotrimeric G protein in order to activate the formation of filopodia, ruffles and stress fibers, respectively (as noted above, this activation is presumably mediated by Cdc42, Rac and Rho). EGF and platelet-derived growth factor (PDGF)
In this review, I highlight recent progress made in identifying components of these signaling pathways, and then raise some concerns and questions for the future. Upstream elements signal transduction
of Rho family mediated pathways
Signal blmsductlon end actin filament organization Zigmond
67
F~um 1
/
F-actin )
~
( ~
?,
~> GEF Cdc42 GAP ? ,
~> Filopodia
<~ (.-.~GTP--.hiv ~ ~::~:::::::::~#? I~>(e.g. GEE RaC GAP~> Tiarnl) (e.g.o~chi maerii n)II
F'actinc:::::~ > Lamellipo
(~C
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"--GDP*~) ~ ' ~ Serpentinereceptor Tyrosinekinasereceptor --~ Phosphorylation (~ 1996 Current Opinion in Cell Biology
This diagram illustrates proposed relationships between receptors, Rho family members, and the cytoskeleton. The data illustrated are primarily obtained from studies in Swiss 3T3 cells. The extracellular ligands are bradykinin (BD), bombesin (BO), PDGF, PMA, and LPA. The receptors for extracellular ligands are serpentine receptors linked to heterotrimeric G proteins, tyrosine kinase receptors, or protein kinase C (C kinase). The kinases are PI3K (which was identified by sensitivity to wortmannin), C kinase (the protein kinase C target of PMA), and Y kinase (the tyrosine kinases, which were identified by sensitivityto tyrphostin or genistein). GTP and GDP refer to the nucleotides bound by the Rho family G proteins.
bind to tyrosine kinase receptors, and PMA binds to a protein kinase C, in order to activate ruffles [3"'1 (see Fig. 1). Between the initial receptor and the Rho family member in the signal transduction pathway, specific kinases may be required. For example, phosphatidylinositol 3-kinase (PI3K) is required for the activation of Rac by the binding of agonists to tyrosine receptors [8°,12,13°,14,15"]. However, PI3K is not required by agonists that induce ruffling via heterotrimeric G proteins, nor is it required for induction of ruffling by PMA [14,16,17°,18"]. A
tyrphostin-25 sensitive kinase is required for activation of Rho by LPA [10,14,19]. Immediately upstream of each Rho family member, a guanine nucleotide exchange factor (GEF) is apparently needed [20,2I°',22"]. The family of GEFs for Rho family members share common motifs, namely a Dbl homology region, which has GEF activity, and a pleckstrin homology domain, which can bind phosphatidylinositol 4,5-bisphosphate (PIP21 and the I~ subunits of heterotrimeric G proteins [23,24,25",26,27°]! A GEF can have specificity for a particular member of the Rho family. Thus, transfection
68
Cytoskeleton
of fibroblasts with Tiaml, a GEF for Rac (and Cdc42), stimulates membrane ruffling, presumably by activating Rac [24].
Downstream elements of Rho family mediated signal transduction pathways Thus far, the downstream elements of pathways that regulate cytoskeletal organization have not been defined. T h e list of activities stimulated by Cdc42, Rac and Rho is long, and includes cascades of kinases that regulate gene transcription and cell growth [4",25°,28-30]. But none of these activities have been linked to Factin rearrangements. Cdc42 and Rac directly activate serine/threonine kinases of the p65 PAK family (kinases homologous to STE20 of yeast and p120 ACK of rats) [31,32°,33]. However, in neutrophils, inhibition of PI3K with wortmannin inhibits chemoattractant activation of p65 PAK and NADPH oxidase, but does not inhibit membrane ruffling [32°]. Thus, activation of this particular PAK is not needed for membrane ruffling. A tyrosine kinase appears to be required downstream of Rho for the formation of stress fibers, as Rho-mediated induction of stress fiber formation in Swiss 3T3 cells is inhibited by the tyrosine kinase inhibitor genistein [6°]. Also interacting with the Rho family are proteins which can negatively regulate their activity by increasing the hydrolysis of their bound GTP; these negative regulators are the GTPase-activating proteins or GAPs [21°*,34°]. In vitro, GAPs show specificity for particular members of the Rho family and hence may regulate specific cytoskeletal patterns [34°-36°]. But the role of these proteins in vivo remains obscure. Injection of a fragment of the Bcr protein which possesses GAP activity for Rac inhibits membrane ruffling induced by P D G F [37]. However, bcrnull mice lack detectable defects in either basal or PDGF-stimulated membrane ruffling, or in F-actin staining [35°]. Thus, the GAP activity of Bcr either does not regulate these F-actin patterns or is functionally redundant with other GAPS. For example, transformation of NIH 3T3 cells with (x chimaerin, another Rac GAP, inhibits the formation of stress fibers induced by LPA [38], a function originally ascribed to Rho in Swiss 3T3 cells. Rho family regulation of phosphatidylinositol (PI) metabolism is of particular interest because increases in PI turnover (but not necessarily in total PIP 2 levels) often correlate with increases in F-actin levels [15°,39°,40,41,42",43°]. However, this story remains fragmentary. As noted above, in some cases Rac activation is dependent on PI3K. In some cases, PI3K may also be activated downstream of Rho family activation [44,45]. Other PI kinases may also be stimulated. In permeabilized platelets, Rac, but not Rho, stimulates the incorporation of phosphate into PIP2 [46°°]. In permeabilized 3T3 cells, Rho, but not Rac, stimulates PI 4-phosphate 5-kinase activity [47]. Recent cloning of both PI 4-kinase and PI 4-phosphate 5-kinase should help to clarify the picture [48,49].
Cytoskeletal targets of regulation T h e F-actin patterns described above involve both actin polymerization and actin cross-linking. Polymerization can be stimulated by increasing the availability of free barbed ends, by increasing the availability of monomeric G-actin, and/or by stabilizing F-actin. T h e spatial pattern of F-actin that is induced depends on associated cross-linking proteins. Attention has focused on factors that regulate these processes, and how these factors might, in turn, respond to messages from the Rho family.
Barbed-end availability The major cytoplasmic G-actin-binding proteins selectively support elongation of F-actin at the barbed end [50",51",52]. Therefore, a key factor in determining the level and distribution of F-actin is the availability of free barbed ends, and this availability is an obvious target for regulation. In permeabilized platelets, Rac increases the availability of free barbed ends, as do a peptide agonist of the thrombin receptor and GTPyS. This peptide agonist, GTP3'S, and Rac also appear to stimulate increases in phosphorylation of PI, resulting in the formation of PIPz. T h e increase in the availability of free barbed ends appears to require PIP2, as it is inhibited by a PIP2-binding peptide from gelsolin (see below) [46°']. Although still preliminary, these studies are exciting because they suggest a causal chain from agonist to effector. Agonists can increase free barbed ends by inhibiting barbed end capping proteins, by de novo assembly of monomers in to a filament (i.e. by de novo nucleation), or by cutting existing filaments. T h e barbed end capping proteins gelsolin and 'capping protein' are inhibited by PIP2, and thus PIP 2 could increase barbed ends either by preventing capping of newly created ends, or possibly by removing a capping protein from existing ends [43,46"']. Regulation of other capping proteins may occur. For example, at adhesion sites, barbed-end availability may depend on regulation of the capping protein tensin. Tensin accumulates with cross-linked integrin receptors even in the absence of actin polymerization [53]. Tensin contains Src homology (SH) 2 domains and numerous phosphorylation sites, making it a potential target for signaling pathways [54,55"]. De novo nucleation might be mediated by the multi-
protein complexes that are a common feature of signal transduction pathways [56]. De novo nucleation sites may be difficult to assay as they are probably labile (shown by the fact that removal of agonists rapidly blocks actin polymerization [57]), and disappear as de novo nucleation sites once monomers are added and the nucleation sites become filament ends. Where such nucleation sites are stable, for example in budding yeast, their detection will be easiest. Thus, the F-actin patches in buds of Saccharomyces cerevisiae may be nucleated in a process requiring analogs of both Cdc42 and talin [58"]. In higher eukaryotes also, talin may play a role in de novo actin
Signal transduction and acUn filament organization Zigmond
nucleation, as it is present at the tips of lamellipodia and plays a key role in induction of attachment plaque formation by integrin [59]. New barbed ends created de novo could grow until capped. Interestingly, the capping rate may be slow enough for elongation at a new barbed end to be significant [60"]. Free barbed ends could be created by cutting of existing filaments by cofilin and its relative destrin. Cofilin and destrin are small proteins that both cut filaments and bind monomeric actin. These actions are inhibited by phosphorylation [61,62]. Stimulation of cells with agonists that increase F-actin levels, such as neutrophil-like HL60 cells stimulated with chemoattractants or platelets stimulated with thrombin, induces dephosphorylation (activation) of destrin and/or cofilin [63-65]. Filament cutting, in addition to increasing free barbed ends, would increase the number of pointed ends and thus help achieve the rapid F-actin turnover that occurs during cell migration.
Monomeric acUn availability Although there is little evidence that inactivation of monomer-binding proteins induces actin polymerization, the local concentration of profilin-actin may enhance the rate of polymerization. Thus, the protein VASP (vasodilator-stimulated phosphoprotein), which binds profilin [66",67"], may enhance the movement of the intracellular parasite Listeria monocytogenes by recruiting profilin-actin to barbed ends near the bacterial surface [67",68"]. VASP accumulates at the bacterial surface by binding to a bacterial protein, ActA [69]. T h e mechanism of Listeria's movement may parallel that of lamellipodial extension. Thus, in uninfected cells, VASP concentrates at sites of actin polymerization such as the tips of lamellae and focal adhesions [68"]. VASP is phosphorylated by protein kinase A, which may inhibit its function, as in platelets its phosphorylation correlates with inhibition of platelet aggregation [67"]. F-actin-binding proteins Control of F-actin-binding sites may regulate membranecytoskeletal attachments. Regulation of vinculin may expose a carboxy-terminal tail fragment of vinculin which can bind to F-actin. T h e actin-binding site on this portion of vinculin is unavailable in intact vinculin. In intact vinculin, the F-actin-binding site is covert because it is masked by intramolecular association with vinculin's head domain [70"']. Vinculin contributes to the attachment of actin filaments to integrins at focal adhesions, and is required for stabilization, but not initiation, of filopodia and lameilipodia [71]. Regulation of covert F-actin-binding domains in the carboxy-terminai domains of ezrin and moesin may also mediate attachment of the cytoskeleton to the membrane [72",73",74]. Overexpression of the carboxy-terminal portion of ezrin can induce lamellipodia [75], whereas antisense oligonucleotides of ezrin and its relatives moesin and radixin decrease filopodia and lamellipodia [76]. Ezrin localizes in membrane ruffles
69
induced by EGF [77], and is a substrate for several kinases, including the E G F receptor kinase.
Regulation of signaling by polymerization T h e signaling pathway itself is regulated by actin polymerization, which binds and organizes reactive components. Inhibiting actin polymerization blocks or greatly diminishes the activation of several cytoskeletal components and disrupts their localization ([59,78",79-81,82"], but see [1]). Thus, actin-binding proteins, whose distribution patterns are altered by agonists, may not be regulated themselves but may merely be controlled by binding (or lack of binding) to newly polymerized actin. This may account for the presence of many of the same actin-binding proteins in filopodia, lamellipodia, and stress fibers. However, each pathway stimulates a specific F-actin pattern that must depend on differences in the targets activated. It is probable that specific actin-binding proteins are targets of the signaling pathway, and that they determine the location of polymerization, the dynamic properties of the filaments formed, and cross-linking between different actin filaments and between filaments and the membrane. Q u e s t i o n s for further i n v e s t i g a t i o n A number of comments must be made in conclusion. Firstly, neither the review nor Figure 1 are comprehensive summaries but rather efforts to organize a small fraction of recently published information. Secondly, the proteins of the Rho family may not be the sole regulators of actin patterns. Indeed, membrane ruffling induced by the attachment of Salmonella typhimuHum to a cell is not inhibited by dominant-negative Rac, suggesting that Rac-independent pathways can induce membrane ruffling [83]. Whether the actin rearrangements induced by chemoattractants, ligation of the IgE receptor, or integrin engagement and clustering are mediated by the Rho family is unknown. Thirdly, most of the experiments implicating the Rho family have used injected constitutively active or inactive forms of the proteins. However, injection probably produces abnormal levels, abnormal localization, and abnormal temporal regulation of the Rho proteins. Thus abnormal results may be produced. In fact, the Rho family might not be part of the direct signaling pathway that leads from agonist to cytoskeleton, and might instead be on a parallel pathway that provides substrates for the signaling pathway. For example, if clustering, not synthesis, of PIP z is a key step in the signaling pathway, stimulation of PIP 2 synthesis by Rho family members may simply maintain normal levels of PIP z. Overexpression of activated Rho family members might result in excess PIP z which then might congregate in clusters that drive the signaling pathway. Fourthly, signaling pathways and cytoskeletal patterns differ between cell types. T h e morphological changes
70
Cytoskeleton
induced by different Rho proteins were mostly observed following injection into starved Swiss 3T3 cells. T h e s e cells are unusual in some respects; for example, E G F increases levels stress fibers in Swiss 3T3 cells, but in most cell types it decreases stress fiber levels. Furthermore, the effects of Ras, Rac and Rho on membrane ruffling in these Swiss 3T3 fibroblast cells differ from the effects observed in epithelial cells [84•,85"]. Fifthly, the task of ordering the steps in the full signaling pathway that leads to a particular actin pattern is complicated because all cells contain multiple signaling pathways that affect actin organization, and because the pathways interact [53,76]. For example, attachment to specific substrata can affect responses to soluble agonists, but soluble agonists also affect adhesive properties. T h e Rho family may be an integration site for signals from substratum and solution. Sixthly, PI metabolism may well be a key link between the Rho family and F-actin patterns, but studies are hampered by inadequate tools. We need ways to measure not only total cellular PIP 2 levels but also local concentrations. T h e high charge of PIP 2 is also a problem because, in purified systems, it can cause PIP 2 to bind to proteins to which it may not have access in vivo. T h e important conclusion that Rac acts through PIP 2 to free barbed ends is based upon on the ability of the gelsolin peptide to block this process. But this domain of gelsolin may bind to cellular components other than PIP z (including F-actin [85•]). Furthermore, even if the peptide does bind to PIP z, the ultimate effects may result from inhibition of other proteins affected by PIP 2, including different actin-binding proteins, phospholipases C or D, or receptor kinases [43",87--89]. Finally, the morphological changes assayed are complex. For example, membrane ruffling requires membrane protrusion in addition to changes in F-actin levels and organization. So far, changes in F-actin levels and F-actin organization have rarely been differentiated; yet they can clearly be independently regulated. Furthermore, the relative amounts of F-actin in stress fibers, membrane ruffles, or diffused throughout the cytoplasm differ between cells. Thus, inhibition of ruffling does not necessarily imply inhibition of net actin polymerization. Indeed, in rat basophilic leukemic cells, wortmannin inhibits FceR1 receptor induced ruffling without inhibiting cell spreading or actin polymerization [90"]. T h e amount of information regarding the pathways from receptor occupancy to actin polymerization and organization is growing explosively. But final understanding is still a long way off.
Acknowledgements Thanks to S Shattil and P Devreotes for helpful discussions, and to L Brass, L Cassimeris, V Nachmias, G Smith, P Sterling and M Symons for reading
drafts of the review. I am supported by the National Institutes of Health (NIH grant AII9883).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.
of special interest of outstanding interest Nobes C, Hall A: Regulation and function of the Rho subfamily of small GTPases. Curt Opin Genet Dev 1994, 4(suppl 1):77-81.
2, Ridley AJ: Membrane ruffling and signal transductlon. Bioessays •e 1994, 16(suppl 5):321-327. Excellent critical review of the signal transduction pathways involved in membrane ruffling.
3. •e
Nobes CD, Hall A: Rho, Ra¢, and Cd¢42 GTPeses regulate the assembly of multlmolecular focal complexes associated with acUn stress fibers, lamelltpodia, and filopodia. Cell 1995, 81:53-62. The authors of this paper describe the induction of filopodia by injection of an activated form of Cdc42. The authors also show that Cdc42 and Rac can induce small attachment plaques in a Rho-independent manner. 4. •=
Kozma R, Ahmed S, Best A, Lim L: The ras-related protein Cdc42Hs and bradyklnln promote formation of peripheral actln mlcrospikes and filopodla in Swiss 3T3 fibroblests. Mol Cell Bio11995, 15:1942-1952. Significantly, the ability of bradykinin to induce filopodia was inhibited by prior injection of dominant-negative Cdc42HsTM7N 5. Cliu R-G, Chen J, Kirn D, McCormick F, Symons M: An essential • role for Rac In Ras transformation. Nature 1995, 374:457-459. Confirms morphological effects of Rac in surface ruffling; shows role for Rac in oncogenic transformation. 6. •
Ridley AJ, Hall A: Signal transduction pathways regulating Rhomediated stress fiber formation: requirement for a tyrosine kinase. EMBO J 1994, 13(suppl 11 ):2600-2610. Shows that the tyroalne kinase inhibitor genistein inhibits the formation of stress fibers induced by extracellular factors or microinjected Rho protein. Genistein also inhibits the Rho-dependent increase in tyrosine phosphorylation of pp125FAK (a phosphorylated attachment plaque protein). 7.
RankinS, Marii N, Narumiya S, Rozengurt E: Botullnum C3 exoenzyme blocks the tyrosine phosphorylstlon of p125FAK and paxlllln induced by bombesln and endothelin. FEBS Lett 1994, 354:315-319.
8. •
Hawkins PT, Equinoa A, Oiu R-Q, Stokoe D, Cooke F, Waiters R, Wennstrom S, Claesson-Welsh L, Evans T, Symons M, Stephens L: PDGF stimulates an Increase in GTP-Rac via activation of phospholnositlde 3-klnase. Curt Biol 1995, 5:393-403. Shows that platelet-derived growth factor (PDGF) stimulates binding of GTP by Rac, and that this activation depends on phosphatidylinositol 3-kinase (PI3K). 9.
Phillips MR, Fsoktistov A, Pillinger MH, Abramson SB: Translocation of p21rac2 from cytosol to plasma membrane is neither necessary for sufficient for neutrophil NADPH oxidase activity. J Bio/Chem 1995, 270:11514-11521.
10.
Peppelenbosch MP, Qui R-G, Vries-Smits Ammd, Tertoolen LGJ, de Last SW, McCormick F, Hall A, Symons MH, Bos JL: Rac mediates growth factor-induced arachidonic acid release. Cell 1995, 81:849-856.
t 1.
Peppelenbosch MP, Tertoolen LG, Hage WJ, de Laat SW: Epidermal growth factor-induced actln remodeling is regulated by 5-1ipoxygenase and cyclooxygenase products. Cell 1993, 74(suppl 3):565-575.
12.
Wennstrom S, Siegbahn A, Yokte K, Arvidsson A, Heldin C, Mori S, Claasson-Welch L: Membrane ruffling and chemotaxis tranduced by the PDGF I~-receptor require the binding site for phosphatidyllnositol-3' kinasa. Oncogene 1994, 9:651-660.
13. Pawson T: Protein modules and signalling networks. Nature • 1995, 373:573-580. Concise review of how tyrosine kinase receptors activate various enzymes. 14.
Nobes CD, Hawkins P, Stevens L, Hall A: Activation of the small GTP-blnding proteins rho and rac by growth factor receptors. J Cell Sci 1995, 108:225-233.
Signal transduction and a d i n filament organization Zigmond
15. Divecha N, Irvine RF: Phospholipld signaling. Cell 1995, • 80:269-278. Critical review of phosphatidylinositol (PI) turnover.
16.
Kovacaovica TJ, Bachelot C, Toker A, Vlehos C J, Duckworth B, Cantley LC, Hartwig JH: I:)hospholnosiUde 3-klnase Inhibition spares actin assembly In activating platelats but reverses plat, let aggregation. J Biol Chem 1995, 270:11358-11366.
17. •
Vlahos C J, Matter WF, Brown RF, Traynor-Kaplan AE, Heyworth RG, Prossnitz ER, Ye RD, Marder P, Scha1,4, Rothfuss KJ et el.: Investigation of neutrophll signal transductlon unslng a specific inhibitor of phosphetidylinositol 3-klnase. J Immunol 1995, 154:2413-2422. Shows that inhibition of PI3K does not inhibit chomoattraotant-induced actin polymerization. 18. Ding J, Vlahos C J, Liu R, Brown RF, Badwey JA: Antagonists • of phosphatidyllnositol 3-kinase block activation of several novel protein Idnases in neutrophils. J Biol Chem 1995, 270:11584-11691. inhibition of PI3K blocks several kinases. 19. Moot,near WH: Lysophosphatidlc acid, • multifunctlonal phosphollpld messenger. J Biol Chem 1995, 270:12949-12952. 20. Hall A: Signal transductlon through smell GTpsses-a tale of two GAPs. Cell 1992, 69:369-391. 21. Hall A: Small GTP-bindlng proteins and the regulation of the •• actln cytoskeloton. Annu Rev Cell Bio11994, 10:31-54. Comprehensive review of GTP-binding proteins. Discusses the apparent requirement for a guanine nucleotide exchange factor (GEF) immediately upstream of the Rho proteins in signal transduction pathways which promote actin filament re-organization. Also discusses GTPase-sctivating proteins (GAPs). 22. Bokoch GM, Bohl BP, Chuang T-H: Guanine nudeotlde • exchange regulates membrane transiocatton of Rec/Rho GTPbinding proteins. J Biol Chem 1994, 269:31674-31679. Shows that release of guanine nucleotide dissociation inhibitor (GDI) is not sufficient to facilitate the binding of GTP to Rac. 23. Habets GGM, Scholes EHM, Zuydgeest D, Kammen RAVD, Stem JC, Barns A, Collard JG: Identification of an invasion-Inducing gene Tlam-1, that encodes a protein with homology to GDPGTP exchangers for Rho-like proteins. Cell 1994, 77:537-549. 24.
Michiels F, Habets GG, Stare JC, Kammen RAvd, Collard JG: A role for Rac in Tiara1-Induced membrane ruffling and invasion. Nature 1995, 375:338-340.
25. •
Symons M: The Rac and Rho pathways as a source of drug targets for Ras-medloted malignancy. Curr Opin Biotechnol 1995, 6:668-674. Review of the transforming properties of the Rho family proteins. 26.
Zheng Y, Olson MF, Hall A, Cerione RA, Toksoz D: Direct involvement of the small GTP-blndln9 protein Rho in Ibc oncogene function. J Biol Chem 1995, 270:9031-9034.
are targets for Cdc42Hs and Racl In neutrophlls. J Biol Chem 1995, 270:10717-10722. 34.
Lamarohe N, Hall A: GAPs for rho-releted GTPsses. Trends Genet 1994: 10:346-440. Review of Rho GAPs. 35. •
Voncken JW, Schalck HV, Kaartinen V, Deemer K, Coates T, Landing B, Pattengal P, Dirseyuk O, Bokcoh GM, Groffen J, Heisterkamp N: Increased rmutrophil respiratory burst in bcrnull mutants. Cell 1995, 80:719-728. A mouse that was homozygous null for Bcr, which is a GAP for Rac, showed a prolonged respiratory burst but no detectable defects in neubophil migration or PDGF-stimulated actin changes in fibmblasts. 36. •
Reinhard J, Scheel AA, Dlekmann D, Hall A, Ruppert C, Bahler M: A novel type of myosin Implicated In signalling by rho family of GTPsses. EMBO J 1995, 14:697-704. A glutathione S-transferase (GST) fusion protein, which includes the GAP homology region from a novel myosin, has GAP activity for RhoA and Cdc42Hs and, to a lesser extent, for Racl. 3?.
Ridley AJ, Self AJ, I.as OF, Paterson HF, Hall A, Marshall C J, Ellis C: Rho family GTPese activating proteins p190, bcr and rhoGAP show distinct spedfk:Rlos in vitro and in vivo. EMBO J 1993, 12:5151-5160.
38.
Herrera R, Shivers BC: Expression of alpha 1-chlmaedn (rsc1 GAP) altars the cytoskelefal and adhesive properties of fibroblast¢ J Cell Biochem 1994, 56(suppl 4):582-591.
39. •
Apgar JR: Polymerization of actln in RBL calls can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used. Mol Biol Cell 1994, 5:313-322. Actin polymerization correlates with the synthesis of phosphatidlyinositol phospate (PIP) and PIP2. 40.
McManee HM, Ingbar DE, Schwartz MA: Adhesion to fibronecttn sUmulatas Inositol lipid synthesis and enhances POGF-Inducad Inosltol lipid breakdown. J Cell Bio/1992, 121:673-678.
41.
Apgar JR: Activation of protein klnase C in rat basophlllc leukemia cells stimulates Increased production of phosphefldylinositol 4-phosphate and phosphhatidyllnositol 4,5-blsphosphate: correlation with actin polymerization. Mo/ Biol Cell 1995, 6:97-108.
42. •
Gips SJ, Kandzari DE, Goldschmidt-Clermont PJ: Growth factor receptors, phosphollpases, phospholipid kinases and acttn organization. Semin Cell Biol 1994, 5:201-208. Interesting review of signaling to the sctin cytoskeleton. 43. •
Janmey PA: Phosphoinosldtides and calcium as regulators of cellular actin assembly end disassembly. Annu Rev Physiol 1994, 56:169-191. Comprehensive review of the effects of PIP2 on the cytoskeleton. 44.
Zhang J, Zhang J, Benovio JL, Sugal M, Wetzker R, Gout I, Rittenhouse SE: Sequestration of a G-protein by I~ subunit or ADP-rlbosylaUon of Rho can inhibit thrombin-inducad activation of plat,fat phosphoinositlde 3-kinase. J Biol Chem 1995, 270:6589-6594.
45.
Tolias KF, Cantley LC, Carpenter CL: Rho family GTPases bind to phosphoinositide klnases. J Biol Chem 1995, 270:17656-17659.
27. •
Harlan JE, Hajduk PJ, Yoon HS, Fesik SW: PIeckstrln homology domains bind to phosphatldyllnosltol-4,5-blsphosphate. Nature 1994, 371:168-170. The binding of pleckstrin to PIP2 is quite weak, having a Kd of 30 pro. 28.
29.
30.
Minden A, Lin A, Claret F-X, Abo A, Karin M: Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPsses Rac and Cdc42Hs. Ceil 1995, 81:1147-1157. Coso OA, Cjoaroe M, Yu J-C, Teramot H, Crsspo P, Xu N, Miki T, Gutkind JS: The small GTP-binding proteins Recl and Cdc42 regulate the activity of JNK/SAPK signaling pathway. Cell 1995, 81:1137-1146. Vojtek AB, Cooper JA: Rho family members: activators of MAP klnase cascades. Cell 1995, 82:527-529.
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46. • ,,
Hartwig JH, Bokoch GM, Carpenter CL, Janmey PA, Taylor I_A, Toker A, Stossel TP: Thrombln receptor Ilgstion end activated Rac uncap act.in filament barbed ends through phospholnositlde synthesis in permeabllized human plat,lets. Cell 1995, 82:643-653. Proposes a possible causal signal transduction pathway from Rac to actin. 47.
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50. •
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31.
33.
Prigmore E, Ahmed S, Best A, Kozma R, Manser E, Segal AW, Lim L: A 68-kDe klnese and NADPH oxidate component p67phox
71
72
Cytoskeleton
GTPTS induces aclin polymerizationin ~ i l i z e d the availability of free barbed ends.
neutrophils by increasing
51. Sun H-Q, Kwiatkowska K, Yin HL: Actln monomer binding • proteins. Curt Opin Cell Biol 1995, 7:102-110. Review of the role of monomer-binding proteins in the regulation of F-actin levels. 52.
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55. •
Lo AH, Janmey PA, Hatwig JH, Chen LB: Interaction of tensin with actin and identification of its three distinct actin-blnding domains. J Cell Big/1994, 125:1067-1075. Shows that incomplete binding by tensin probably accounts for what was previously thought to be leaky capping by the tensin fragment insertin. When tensin concentrations are insufficient to cause complete capping, elongation of F-actin is slowed but not blocked. 56.
Schwartz MA, Luna EJ: How actln binds and assembles onto plasma membranes from Dictyostelium discoideum. J Cell Biol 1988, 107:201-209.
57.
Cassimeris L, McNeill H, Zigmond SH: Chemoattractant stimulated neutrophils contain two populations of actln filaments that differ in their spatial distributions and relative stabilities. J Cell Biol 1990, 110:1067-1075.
58. •
Li R, Zheng Y, Drubin DG: Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast. J Cell Biol 1995, 128:599-615. On the basis of several experiments in permeabilized yeast, the authors propose that actin is nucleated from a component(s) other than an existing actin filament. The process requires analogs of both Cdc42 and talin. 59.
Chen H-C, Appenddu PA, Parsons JT, Hildebrand JD, Schaller MD, Guan J-L: Interaction of focal adhesion kinase with cytoskeletal protein talin. J Biol Chem 1995, 270:16995-16999.
DiNubile MJ, Cassimeris LU, Joyce M, Zigmond SH: Actin filament • barbed-end cappng activity in neutrophil lysetes: the role of CapZ. Mol Biol Cell 1995, 12:1659-1671. Kinetics of the capping protein 1~2, a non-muscle analog of Cap Z, are relatively slow, thus potentially allowing elongation of actin filaments before capping. 61. Morgan TE, Lockerbie RO, Minamide LS, Browing MD, Bamberg JR: Isolation and characterization of a regulated form of actin depolymerizing factor. J Cell Bio11993, 122:623-633.
Listeria monocytogenes and Listeria ivanovii to the actln-based cytoskeleton of mammalian cells. EMBO J 1995, 14:101-108. Describes the cellular localization of VASP, and discusses its role in bacterial cell movement. Also discusses the function of VASP in mammalian cells such as platelets. 69.
70. =.
Johnson RP, Craig SW: F-ectln binding site masked by the intramolecular association of vlnculin head and tall domains. Nature 1995, 373:261-264. Elegant study showing that vinculin has an actin-binding site, which is normally blocked by another portion of the vinculin protein. 71.
Agnew BJ, Minamide LS, Bamberg JR: Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. J Biol Chem 1995, 170:17582-17587.
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KanamoriT, Hayakawa T, Suzuki M, Titani K: Identification of two 17-kDa rat parotid gland phosphoproteins, subjects for dephosphorylation upon 13-adrenergic stimulation, as destrinand cofilln-like proteins. J Biol Chem 1995, 270:8061-8067.
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SuzukiK, Yamaguchi T, Tanaka T, Kawanishi T, Nishimaki T, Morami T, Yamamoto K, Tsuji T, Irimura T, Hayakawa T, Takahasi A: Activation induces dephosphorylation of cofilin and its translocation to the plasma membranes in neutrophiHike differentiated HL-60 cells. J Biol Chem 1995, 270:19551-19556. Davidson MML, Haslam PJ: Dephosphorylation of cofilin in stimulated plat•lets: roles for a GTP-bindng protein and Ca 2+. Biochem J, 1994, 301:41-47.
Varnum-Finney B, Reichart LF: Vinculln-deficlent PC12 cell lines extend unstable lamelllpodia and filopodla and have • reduced rate of neurlte outgrowth. J Cell Biol 1994, 127:1071-1084.
72.
TurunenO, Wahlstrom T, Vaheri A: Ezrin has a COOH-termlnal actin-blndlng site that is conserved In the ezrtn protein family. J Cell Bio11995, 126:1445-1453. A GST fusion protein of ezrin was attached to a glutathione column. When cytoplasm was passed over the column, actin and other cytoplasmic proteins were retained. •
73. •
Pestonjamasp K, Amieva M, Strassel CP, Nauseef WM, Furthmayr H, Luna F_J:Moesin, ezrln and p205 are actin-blndlng proteins associated with neutrophll plasma membranes. Mol Biol Cell 1995, 6:247-259. Shows that moesin, ezrin and a 205 kDa protein present in neutrophil membranes all bind F-actin in a blot overlay using t 251.F.actin. The actin-binding site in moesin was localized to the extreme carboxyl terminus. 74.
Gary R, Bretsher A: Ezrln self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that Includes the F-actln binding site. Mol Biol Cell 1995, 6:1061-1075.
75.
Martin M, Andreoli C, Sahuquet A, Montcourrier P, Algrain M, Mangeat P: Ezrln NH2-termlnal domain Inhibits the cell extension activity of the COOH-terminal domain. J Cell Biol 1995, 128:1081-1093.
76.
Takeuchi K, Sato N, Kasehara H, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S: Perturbation of cell adhesion and microvilll formation by antlsense oligonucleotides to ERM family members. J Cell Bio11994, 108:2369-2382.
77.
Bretscher, A: Rapid phosphorylation and reorganization of ezrin end spectrln accompany morphological changes Induced in A-431 cells by epidermal growth factor. J Cell Biol 1989, 108:921-930.
60.
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Smith CA, Portnoy DA, Theriot JA: Asymmetric distribution of the List•tie monocytogenes ACtA protein Is required end sufficient to direct actin-besed motility. Mol Microbio/1995, in press.
78. Shattil S J, Ginsberg MH, Brugge JS: Adhesive signaling in • platelets. Curr Opin Cell Biol 1994, 6:695-704. Review of inside-out and outside-in signaling in platelets. In inside-out signaling, a soluble mediator affects cell adhesion. In outside-in signaling, adhesion affects cell function. Also discusses the fact that inhibition of actin polymerization blocks or greatly diminishes the activation of several cytoskeletal components and disrupts their localization. 79.
Fujii C, Yanagi S, Sada K, Nagai K, Taniguchi T, Yamamura H: Involvement of protein-tyrosine kinase p72syk in collageninduced signal transductlon in platelets. Eur J Biochem 1994, 226(suppl 1):243-248.
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Lin TH, Rosales C, Mondal K, Haskill JBS, Juliano RL: Integrinmediated tyroslne phosphorylation and cytoklne message induction in monocytic cells: a possible signaling role for the Syk kinase. J Biol Chem 1995, 270:16189-16197.
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Chen Q, Kinch MS, Lin TH, Burride K, Juliano RL: Integrinmediated cell adhesion activates mitogen-actlveted protein kinases. J Biol Chem 1994, 269:26602-26605.
66. ••
82. •
67. •
83.
Jones BD, Paterson HF, Hall A, Falkow S: Salmonella typhimurium induces membrane ruffling by a growth factor receptor independent mechanism. Proc Natl Acad Sci USA 1993, 90:10390-10394.
84. •
Ridley A, Comoglio PM, Hall A: Regulation of scatter factor/hepatocyte growth factor reponses by Ras, Rac, and Rho in MDCK cells. Mo/Cell Big/1995, 15:1110-1122.
ReinhardM, Giehl K, Abel K, Haffner C, Jarchau T, Hoppa V: The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilin. EMBO J 1995, 14:1583-1589. Vasodilator-stimulated phosphoprotein (VASP) is the first protein, other than actin, that has been shown to bind to profilin. Pistor S, Chakraborty T, Walter U, Wehland J: The bacterial actin nucletor protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins. Curt Biol 1995, 5:12-21. Review of the properties of ActA and VASP. 68. Chakraborty T, Ebel F, Domann E, Niebuhr K, Gerstel B, Pistor S, • Temm-GroveCJ, Jockusch BM, Reinhard M, Walter U, Wehland J: A focal adhesion factor directly linking intracellulsrly motile
Bachman ES, McClay DR: Characterization of moesin in the sea urchin Lytechinus variegetus: redistribution to the plasma membrane following fertilization is inhibited by cytochalesin B. J Cell Sci 1995, 108:161-171. The sea urchin moesin molecule shares 75% amino acid similarity with the amino terminus of vertebrate moesin.
Signal transduction and actin filament organization Zigmond
In MDCK cells, dominant-negative Rac inhibited ruffling induced by scatter factor or Ras. However, injection of active Rac did not induce ruffling, injection of active Ras stimulated spreading and ruffling but not motility. See also Niehiyama et al. [84"]. 85. •
Nishiyama T, Sasaki T, Takalshi K, Kato M, Yaku H, Araki K, Matsuura Y, Takai Y: Rec p21 is involved in insulin-induced membrane ruffling and rho p21 Is involved In hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acefate (TPA)-Induced membrane ruffling In KB cells. Mol Cell Biol 1994, 14:2447-2458. Shows that a different morphology of ruffles is induced by insulin, scatter factor or 19-O-tetradecanoylphorbol-13-acetate(TPA). Injection of either GTP~-Rac or GTPI~-Rho also induced ruffling but the form of the ruffles differed. Injection of C3 transferase inhibited ruffling induced by scatter factor. See also Ridley et el. [83"]. 86.
Janmey PA, Lamb J, Allen PG, Matsudalra PT: Phospholnosltidebinding peptldes derived from the sequences of gelsolin and villin. J Biol Chem 1992, 267:11818-11823.
73
8'7.
Abrams CS, Wu H, Zhac W, Belmonte E, White D, Brass LF:Pleckslxin Inhibits phosphoinosltJde hydrolysis Initiated by G-protein-coupled and growth factor receptors. J Biol Chem 1995, 270:14485-14492.
88.
Onorato JJ, Gillis ME, Liu Y, Benovic JL, Ruoho AE: The 13-adranergic receptor klnese (GRK2) Is regulated by phospholiplds- J B/o/Chem 1995, 270:21346-21353.
89.
TeruiT, Kahn RA, Randazzo PA: Effects of acid phospholipids on nucleotide exchange properties of ADP-dbosylatlon factor 1. J Biol Chem 1994, 269:28130-28135.
90. •
Barker SA, Caldwell KK, Hall A, Martinez AM, Pfeiffer JR, Oliver JM, Wilson BS: Wortmannln blocks lipid and protein klnase activities assolcated with PI 3-kinese and inhibits a subset of responses induced by FceR1 cross-linking, Mol Biol Cell 1995, 6:1145-1158. Describes an interesting sorting of the activities affected by wortmannin.
Signal transduction and actin filament organization Sally H Zigmond Small GTP-binding proteins of the Rho family appear to integrate extracellular signals from diverse receptor types and initiate rearrangements of F-actin. Active members of the Rho family, Rho and Rac, are now joined by Cdc42 which induces filopodia. Downstream of the Rho family proteins, actin polymerization may be induced by an increase in the availability of actin filament barbed ends. Actin organization may be affected by exposure of actin-binding sites on proteins such as vinculin and ezrin.
Address Biology Department, Universityof Pennsylvania, Philadelphia, PA 19104-6018, USA; e-mail: [email protected] Current Opinion in Cell Biology 1996, 8:66-73 © Current Biology Ltd ISSN 0955-0674 Abbreviations EGF epidermal growth factor GTPase-activating protein GAP GEF guanine nucleotide exchange factor LPA lysophosphatidic acid PAK protein-activated kinase PDGF platelet-derived growth factor PI phosphatidylinositol Pl3K phosphatidylinositol 3-kinase phosphatidylinositol 4,5-bisphosphate PIP2 PMA phorbol myristic acetate VASP vasodilator-stimulated phosphoprotein
by the dominant-negative (i.e. GDP-bound form) of the appropriate Rho family member. Thus, injection of dominant-negative Cdc42 blocks the formation of filopodia induced by bradykinin [3°°,4"°], and dominant-negative Rac blocks the formation of ruffles induced by epidermal growth factor (EGF), phorbol myristic acetate (PMA), or bombesin [2",5°]. Inactivation of Rho with the C 3 transferase from Clostridium botulinum blocks the formation of stress fibers induced by lysophosphatidic acid (LPA) [1,6",7]. T h e data suggest that Rho family members mediate these morphological changes that are induced by extracellular agonists. In some cases, the extracellular agonist has also been shown to alter the subcellular location of a Rho family member and/or to stimulate G T P exchange for GDP. However, these changes are usually small [8°,9]. In addition to inducing these specific changes, all members of the Rho family share some functions, and there is cross-talk between family members. Thus, each member of the Rho family can can induce attachment plaques, which are defined by the presence of paxillin and vinculin, two cytoskeletal proteins [3°°]. A given agonist can activate more than one member of the Rho family, and one member of the family can activate another. Thus, Cdc42 can activate Rac, and Rac can activate Rho (see Fig. 1). T h e latter pathway requires the activity of phospholipase A2 and lipoxygenase, presumably to form a leukotriene intermediate [10,11].
Introduction
In response to environmental signals, a cell changes both its shape and its degree of attachment to the substratum. These changes are caused, at least in part, by rearrangements of the actin cytoskeleton. If assembled in long bundles, F-actin supports finger-like protrusions of the plasma membrane known as filopodia; if assembled as a meshwork, it supports sheet-like protrusions known as ruffles or lamellipodia; if present in bundles coupled to attachment plaques, actin 'stress fibers' exert force against the substratum. T h e signals that trigger these different cytoskeletal patterns act through diverse receptors, but it now appears that the pathways leading from these receptors converge on one or more members of the Rho family of small GTP-binding proteins. These proteins are Cdc42, Rac and Rho [1,2"°].
Upstream of the Rho family, only a few elements have been defined. Downstream pathways activate many kinases and transcription factors, and promote cell growth, but it is not known which, if any, of these activities affects the cytoskeleton. However, some pathway eventually stimulates actin polymerization at the barbed (high affinity) end of actin filaments, and some pathway also modulates the association of actin-binding proteins with actin [1,2°°].
Remarkably, when injected in a constitutively active (i.e. a GTP-bound) form into Swiss 3T3 fibroblasts, each of these Rho family members induces unique morphological changes that involve rearrangement of F-actin. Constitutively active Cdc42 induces filopodia [3°'], Rac induces membrane ruffles, and Rho induces stress fibers. When these same morphological changes are induced by extracellular agonists, they can be blocked
Pathways leading to the cytoskeletal changes outlined above are initiated by agonists that bind to a variety of receptors. Bradykinin, bombesin and LPA each bind to a serpentine-type receptor that is linked to a heterotrimeric G protein in order to activate the formation of filopodia, ruffles and stress fibers, respectively (as noted above, this activation is presumably mediated by Cdc42, Rac and Rho). EGF and platelet-derived growth factor (PDGF)
In this review, I highlight recent progress made in identifying components of these signaling pathways, and then raise some concerns and questions for the future. Upstream elements signal transduction
of Rho family mediated pathways
Signal blmsductlon end actin filament organization Zigmond
67
F~um 1
/
F-actin )
~
( ~
?,
~> GEF Cdc42 GAP ? ,
~> Filopodia
<~ (.-.~GTP--.hiv ~ ~::~:::::::::~#? I~>(e.g. GEE RaC GAP~> Tiarnl) (e.g.o~chi maerii n)II
F'actinc:::::~ > Lamellipo
(~C
~L_GDP
kinase
"
7 Y kinase
)
(--~GTP--~ •
Rho
~ '
•
<~ Stressfibers
"--GDP*~) ~ ' ~ Serpentinereceptor Tyrosinekinasereceptor --~ Phosphorylation (~ 1996 Current Opinion in Cell Biology
This diagram illustrates proposed relationships between receptors, Rho family members, and the cytoskeleton. The data illustrated are primarily obtained from studies in Swiss 3T3 cells. The extracellular ligands are bradykinin (BD), bombesin (BO), PDGF, PMA, and LPA. The receptors for extracellular ligands are serpentine receptors linked to heterotrimeric G proteins, tyrosine kinase receptors, or protein kinase C (C kinase). The kinases are PI3K (which was identified by sensitivity to wortmannin), C kinase (the protein kinase C target of PMA), and Y kinase (the tyrosine kinases, which were identified by sensitivityto tyrphostin or genistein). GTP and GDP refer to the nucleotides bound by the Rho family G proteins.
bind to tyrosine kinase receptors, and PMA binds to a protein kinase C, in order to activate ruffles [3"'1 (see Fig. 1). Between the initial receptor and the Rho family member in the signal transduction pathway, specific kinases may be required. For example, phosphatidylinositol 3-kinase (PI3K) is required for the activation of Rac by the binding of agonists to tyrosine receptors [8°,12,13°,14,15"]. However, PI3K is not required by agonists that induce ruffling via heterotrimeric G proteins, nor is it required for induction of ruffling by PMA [14,16,17°,18"]. A
tyrphostin-25 sensitive kinase is required for activation of Rho by LPA [10,14,19]. Immediately upstream of each Rho family member, a guanine nucleotide exchange factor (GEF) is apparently needed [20,2I°',22"]. The family of GEFs for Rho family members share common motifs, namely a Dbl homology region, which has GEF activity, and a pleckstrin homology domain, which can bind phosphatidylinositol 4,5-bisphosphate (PIP21 and the I~ subunits of heterotrimeric G proteins [23,24,25",26,27°]! A GEF can have specificity for a particular member of the Rho family. Thus, transfection
68
Cytoskeleton
of fibroblasts with Tiaml, a GEF for Rac (and Cdc42), stimulates membrane ruffling, presumably by activating Rac [24].
Downstream elements of Rho family mediated signal transduction pathways Thus far, the downstream elements of pathways that regulate cytoskeletal organization have not been defined. T h e list of activities stimulated by Cdc42, Rac and Rho is long, and includes cascades of kinases that regulate gene transcription and cell growth [4",25°,28-30]. But none of these activities have been linked to Factin rearrangements. Cdc42 and Rac directly activate serine/threonine kinases of the p65 PAK family (kinases homologous to STE20 of yeast and p120 ACK of rats) [31,32°,33]. However, in neutrophils, inhibition of PI3K with wortmannin inhibits chemoattractant activation of p65 PAK and NADPH oxidase, but does not inhibit membrane ruffling [32°]. Thus, activation of this particular PAK is not needed for membrane ruffling. A tyrosine kinase appears to be required downstream of Rho for the formation of stress fibers, as Rho-mediated induction of stress fiber formation in Swiss 3T3 cells is inhibited by the tyrosine kinase inhibitor genistein [6°]. Also interacting with the Rho family are proteins which can negatively regulate their activity by increasing the hydrolysis of their bound GTP; these negative regulators are the GTPase-activating proteins or GAPs [21°*,34°]. In vitro, GAPs show specificity for particular members of the Rho family and hence may regulate specific cytoskeletal patterns [34°-36°]. But the role of these proteins in vivo remains obscure. Injection of a fragment of the Bcr protein which possesses GAP activity for Rac inhibits membrane ruffling induced by P D G F [37]. However, bcrnull mice lack detectable defects in either basal or PDGF-stimulated membrane ruffling, or in F-actin staining [35°]. Thus, the GAP activity of Bcr either does not regulate these F-actin patterns or is functionally redundant with other GAPS. For example, transformation of NIH 3T3 cells with (x chimaerin, another Rac GAP, inhibits the formation of stress fibers induced by LPA [38], a function originally ascribed to Rho in Swiss 3T3 cells. Rho family regulation of phosphatidylinositol (PI) metabolism is of particular interest because increases in PI turnover (but not necessarily in total PIP 2 levels) often correlate with increases in F-actin levels [15°,39°,40,41,42",43°]. However, this story remains fragmentary. As noted above, in some cases Rac activation is dependent on PI3K. In some cases, PI3K may also be activated downstream of Rho family activation [44,45]. Other PI kinases may also be stimulated. In permeabilized platelets, Rac, but not Rho, stimulates the incorporation of phosphate into PIP2 [46°°]. In permeabilized 3T3 cells, Rho, but not Rac, stimulates PI 4-phosphate 5-kinase activity [47]. Recent cloning of both PI 4-kinase and PI 4-phosphate 5-kinase should help to clarify the picture [48,49].
Cytoskeletal targets of regulation T h e F-actin patterns described above involve both actin polymerization and actin cross-linking. Polymerization can be stimulated by increasing the availability of free barbed ends, by increasing the availability of monomeric G-actin, and/or by stabilizing F-actin. T h e spatial pattern of F-actin that is induced depends on associated cross-linking proteins. Attention has focused on factors that regulate these processes, and how these factors might, in turn, respond to messages from the Rho family.
Barbed-end availability The major cytoplasmic G-actin-binding proteins selectively support elongation of F-actin at the barbed end [50",51",52]. Therefore, a key factor in determining the level and distribution of F-actin is the availability of free barbed ends, and this availability is an obvious target for regulation. In permeabilized platelets, Rac increases the availability of free barbed ends, as do a peptide agonist of the thrombin receptor and GTPyS. This peptide agonist, GTP3'S, and Rac also appear to stimulate increases in phosphorylation of PI, resulting in the formation of PIPz. T h e increase in the availability of free barbed ends appears to require PIP2, as it is inhibited by a PIP2-binding peptide from gelsolin (see below) [46°']. Although still preliminary, these studies are exciting because they suggest a causal chain from agonist to effector. Agonists can increase free barbed ends by inhibiting barbed end capping proteins, by de novo assembly of monomers in to a filament (i.e. by de novo nucleation), or by cutting existing filaments. T h e barbed end capping proteins gelsolin and 'capping protein' are inhibited by PIP2, and thus PIP 2 could increase barbed ends either by preventing capping of newly created ends, or possibly by removing a capping protein from existing ends [43,46"']. Regulation of other capping proteins may occur. For example, at adhesion sites, barbed-end availability may depend on regulation of the capping protein tensin. Tensin accumulates with cross-linked integrin receptors even in the absence of actin polymerization [53]. Tensin contains Src homology (SH) 2 domains and numerous phosphorylation sites, making it a potential target for signaling pathways [54,55"]. De novo nucleation might be mediated by the multi-
protein complexes that are a common feature of signal transduction pathways [56]. De novo nucleation sites may be difficult to assay as they are probably labile (shown by the fact that removal of agonists rapidly blocks actin polymerization [57]), and disappear as de novo nucleation sites once monomers are added and the nucleation sites become filament ends. Where such nucleation sites are stable, for example in budding yeast, their detection will be easiest. Thus, the F-actin patches in buds of Saccharomyces cerevisiae may be nucleated in a process requiring analogs of both Cdc42 and talin [58"]. In higher eukaryotes also, talin may play a role in de novo actin
Signal transduction and acUn filament organization Zigmond
nucleation, as it is present at the tips of lamellipodia and plays a key role in induction of attachment plaque formation by integrin [59]. New barbed ends created de novo could grow until capped. Interestingly, the capping rate may be slow enough for elongation at a new barbed end to be significant [60"]. Free barbed ends could be created by cutting of existing filaments by cofilin and its relative destrin. Cofilin and destrin are small proteins that both cut filaments and bind monomeric actin. These actions are inhibited by phosphorylation [61,62]. Stimulation of cells with agonists that increase F-actin levels, such as neutrophil-like HL60 cells stimulated with chemoattractants or platelets stimulated with thrombin, induces dephosphorylation (activation) of destrin and/or cofilin [63-65]. Filament cutting, in addition to increasing free barbed ends, would increase the number of pointed ends and thus help achieve the rapid F-actin turnover that occurs during cell migration.
Monomeric acUn availability Although there is little evidence that inactivation of monomer-binding proteins induces actin polymerization, the local concentration of profilin-actin may enhance the rate of polymerization. Thus, the protein VASP (vasodilator-stimulated phosphoprotein), which binds profilin [66",67"], may enhance the movement of the intracellular parasite Listeria monocytogenes by recruiting profilin-actin to barbed ends near the bacterial surface [67",68"]. VASP accumulates at the bacterial surface by binding to a bacterial protein, ActA [69]. T h e mechanism of Listeria's movement may parallel that of lamellipodial extension. Thus, in uninfected cells, VASP concentrates at sites of actin polymerization such as the tips of lamellae and focal adhesions [68"]. VASP is phosphorylated by protein kinase A, which may inhibit its function, as in platelets its phosphorylation correlates with inhibition of platelet aggregation [67"]. F-actin-binding proteins Control of F-actin-binding sites may regulate membranecytoskeletal attachments. Regulation of vinculin may expose a carboxy-terminal tail fragment of vinculin which can bind to F-actin. T h e actin-binding site on this portion of vinculin is unavailable in intact vinculin. In intact vinculin, the F-actin-binding site is covert because it is masked by intramolecular association with vinculin's head domain [70"']. Vinculin contributes to the attachment of actin filaments to integrins at focal adhesions, and is required for stabilization, but not initiation, of filopodia and lameilipodia [71]. Regulation of covert F-actin-binding domains in the carboxy-terminai domains of ezrin and moesin may also mediate attachment of the cytoskeleton to the membrane [72",73",74]. Overexpression of the carboxy-terminal portion of ezrin can induce lamellipodia [75], whereas antisense oligonucleotides of ezrin and its relatives moesin and radixin decrease filopodia and lamellipodia [76]. Ezrin localizes in membrane ruffles
69
induced by EGF [77], and is a substrate for several kinases, including the E G F receptor kinase.
Regulation of signaling by polymerization T h e signaling pathway itself is regulated by actin polymerization, which binds and organizes reactive components. Inhibiting actin polymerization blocks or greatly diminishes the activation of several cytoskeletal components and disrupts their localization ([59,78",79-81,82"], but see [1]). Thus, actin-binding proteins, whose distribution patterns are altered by agonists, may not be regulated themselves but may merely be controlled by binding (or lack of binding) to newly polymerized actin. This may account for the presence of many of the same actin-binding proteins in filopodia, lamellipodia, and stress fibers. However, each pathway stimulates a specific F-actin pattern that must depend on differences in the targets activated. It is probable that specific actin-binding proteins are targets of the signaling pathway, and that they determine the location of polymerization, the dynamic properties of the filaments formed, and cross-linking between different actin filaments and between filaments and the membrane. Q u e s t i o n s for further i n v e s t i g a t i o n A number of comments must be made in conclusion. Firstly, neither the review nor Figure 1 are comprehensive summaries but rather efforts to organize a small fraction of recently published information. Secondly, the proteins of the Rho family may not be the sole regulators of actin patterns. Indeed, membrane ruffling induced by the attachment of Salmonella typhimuHum to a cell is not inhibited by dominant-negative Rac, suggesting that Rac-independent pathways can induce membrane ruffling [83]. Whether the actin rearrangements induced by chemoattractants, ligation of the IgE receptor, or integrin engagement and clustering are mediated by the Rho family is unknown. Thirdly, most of the experiments implicating the Rho family have used injected constitutively active or inactive forms of the proteins. However, injection probably produces abnormal levels, abnormal localization, and abnormal temporal regulation of the Rho proteins. Thus abnormal results may be produced. In fact, the Rho family might not be part of the direct signaling pathway that leads from agonist to cytoskeleton, and might instead be on a parallel pathway that provides substrates for the signaling pathway. For example, if clustering, not synthesis, of PIP z is a key step in the signaling pathway, stimulation of PIP 2 synthesis by Rho family members may simply maintain normal levels of PIP z. Overexpression of activated Rho family members might result in excess PIP z which then might congregate in clusters that drive the signaling pathway. Fourthly, signaling pathways and cytoskeletal patterns differ between cell types. T h e morphological changes
70
Cytoskeleton
induced by different Rho proteins were mostly observed following injection into starved Swiss 3T3 cells. T h e s e cells are unusual in some respects; for example, E G F increases levels stress fibers in Swiss 3T3 cells, but in most cell types it decreases stress fiber levels. Furthermore, the effects of Ras, Rac and Rho on membrane ruffling in these Swiss 3T3 fibroblast cells differ from the effects observed in epithelial cells [84•,85"]. Fifthly, the task of ordering the steps in the full signaling pathway that leads to a particular actin pattern is complicated because all cells contain multiple signaling pathways that affect actin organization, and because the pathways interact [53,76]. For example, attachment to specific substrata can affect responses to soluble agonists, but soluble agonists also affect adhesive properties. T h e Rho family may be an integration site for signals from substratum and solution. Sixthly, PI metabolism may well be a key link between the Rho family and F-actin patterns, but studies are hampered by inadequate tools. We need ways to measure not only total cellular PIP 2 levels but also local concentrations. T h e high charge of PIP 2 is also a problem because, in purified systems, it can cause PIP 2 to bind to proteins to which it may not have access in vivo. T h e important conclusion that Rac acts through PIP 2 to free barbed ends is based upon on the ability of the gelsolin peptide to block this process. But this domain of gelsolin may bind to cellular components other than PIP z (including F-actin [85•]). Furthermore, even if the peptide does bind to PIP z, the ultimate effects may result from inhibition of other proteins affected by PIP 2, including different actin-binding proteins, phospholipases C or D, or receptor kinases [43",87--89]. Finally, the morphological changes assayed are complex. For example, membrane ruffling requires membrane protrusion in addition to changes in F-actin levels and organization. So far, changes in F-actin levels and F-actin organization have rarely been differentiated; yet they can clearly be independently regulated. Furthermore, the relative amounts of F-actin in stress fibers, membrane ruffles, or diffused throughout the cytoplasm differ between cells. Thus, inhibition of ruffling does not necessarily imply inhibition of net actin polymerization. Indeed, in rat basophilic leukemic cells, wortmannin inhibits FceR1 receptor induced ruffling without inhibiting cell spreading or actin polymerization [90"]. T h e amount of information regarding the pathways from receptor occupancy to actin polymerization and organization is growing explosively. But final understanding is still a long way off.
Acknowledgements Thanks to S Shattil and P Devreotes for helpful discussions, and to L Brass, L Cassimeris, V Nachmias, G Smith, P Sterling and M Symons for reading
drafts of the review. I am supported by the National Institutes of Health (NIH grant AII9883).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.
of special interest of outstanding interest Nobes C, Hall A: Regulation and function of the Rho subfamily of small GTPases. Curt Opin Genet Dev 1994, 4(suppl 1):77-81.
2, Ridley AJ: Membrane ruffling and signal transductlon. Bioessays •e 1994, 16(suppl 5):321-327. Excellent critical review of the signal transduction pathways involved in membrane ruffling.
3. •e
Nobes CD, Hall A: Rho, Ra¢, and Cd¢42 GTPeses regulate the assembly of multlmolecular focal complexes associated with acUn stress fibers, lamelltpodia, and filopodia. Cell 1995, 81:53-62. The authors of this paper describe the induction of filopodia by injection of an activated form of Cdc42. The authors also show that Cdc42 and Rac can induce small attachment plaques in a Rho-independent manner. 4. •=
Kozma R, Ahmed S, Best A, Lim L: The ras-related protein Cdc42Hs and bradyklnln promote formation of peripheral actln mlcrospikes and filopodla in Swiss 3T3 fibroblests. Mol Cell Bio11995, 15:1942-1952. Significantly, the ability of bradykinin to induce filopodia was inhibited by prior injection of dominant-negative Cdc42HsTM7N 5. Cliu R-G, Chen J, Kirn D, McCormick F, Symons M: An essential • role for Rac In Ras transformation. Nature 1995, 374:457-459. Confirms morphological effects of Rac in surface ruffling; shows role for Rac in oncogenic transformation. 6. •
Ridley AJ, Hall A: Signal transduction pathways regulating Rhomediated stress fiber formation: requirement for a tyrosine kinase. EMBO J 1994, 13(suppl 11 ):2600-2610. Shows that the tyroalne kinase inhibitor genistein inhibits the formation of stress fibers induced by extracellular factors or microinjected Rho protein. Genistein also inhibits the Rho-dependent increase in tyrosine phosphorylation of pp125FAK (a phosphorylated attachment plaque protein). 7.
RankinS, Marii N, Narumiya S, Rozengurt E: Botullnum C3 exoenzyme blocks the tyrosine phosphorylstlon of p125FAK and paxlllln induced by bombesln and endothelin. FEBS Lett 1994, 354:315-319.
8. •
Hawkins PT, Equinoa A, Oiu R-Q, Stokoe D, Cooke F, Waiters R, Wennstrom S, Claesson-Welsh L, Evans T, Symons M, Stephens L: PDGF stimulates an Increase in GTP-Rac via activation of phospholnositlde 3-klnase. Curt Biol 1995, 5:393-403. Shows that platelet-derived growth factor (PDGF) stimulates binding of GTP by Rac, and that this activation depends on phosphatidylinositol 3-kinase (PI3K). 9.
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46. • ,,
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Signal transduction and actin filament organization Zigmond
In MDCK cells, dominant-negative Rac inhibited ruffling induced by scatter factor or Ras. However, injection of active Rac did not induce ruffling, injection of active Ras stimulated spreading and ruffling but not motility. See also Niehiyama et al. [84"]. 85. •
Nishiyama T, Sasaki T, Takalshi K, Kato M, Yaku H, Araki K, Matsuura Y, Takai Y: Rec p21 is involved in insulin-induced membrane ruffling and rho p21 Is involved In hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acefate (TPA)-Induced membrane ruffling In KB cells. Mol Cell Biol 1994, 14:2447-2458. Shows that a different morphology of ruffles is induced by insulin, scatter factor or 19-O-tetradecanoylphorbol-13-acetate(TPA). Injection of either GTP~-Rac or GTPI~-Rho also induced ruffling but the form of the ruffles differed. Injection of C3 transferase inhibited ruffling induced by scatter factor. See also Ridley et el. [83"]. 86.
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90. •
Barker SA, Caldwell KK, Hall A, Martinez AM, Pfeiffer JR, Oliver JM, Wilson BS: Wortmannln blocks lipid and protein klnase activities assolcated with PI 3-kinese and inhibits a subset of responses induced by FceR1 cross-linking, Mol Biol Cell 1995, 6:1145-1158. Describes an interesting sorting of the activities affected by wortmannin.