23
Integration of signals to the Arp2/3 complex
Alissa M Weaver, Michael E Young, Wei-Lih Lee and John A Cooper The Arp2/3 complex is necessary for nucleating the formation of branched networks of actin ®laments at the cell cortex, and an increasing number of proteins able to activate the Arp2/3 complex have been described. The Wiskott±Aldrich syndrome protein (WASP) family and cortactin comprise the large majority of the known activators. WASPs bind to Arp2/3 via an acidic (A) domain, and a WH2 domain appears to bring an actin monomer to Arp2/3, promoting the nucleation of the new ®lament. Cortactin also binds the Arp2/3 complex via an A domain; however, it also binds to actin ®laments, which helps activate the Arp2/3 complex and stabilise the newly created branches between the ®laments. Addresses Department of Cell Biology, Washington University School of Medicine, St Louis, MO 63130, USA Campus Box 8228, 660 South Euclid Avenue, St Louis, MO 63110, USA e-mail:
[email protected]
Current Opinion in Cell Biology 2003, 15:23±30 This review comes from a themed issue on Cell structure and dynamics Edited by Michel Bornens and Laura M Machesky 0955-0674/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(02)00015-7 Abbreviations A acidic Arp2/3 actin-related protein 2/3 ARPC Arp complex component GEF GDP exchange factor N-WASP neuronal WASP SH3 Src homology 3 TH2/3 tail homology 2/3 WA WH2±acidic WASP Wiskott±Aldrich syndrome protein WH2 WASP homology 2 WIP WASP-interacting protein
Introduction
Many types of cell motility depend on the assembly of actin ®laments, which can drive membrane protrusion and vesicle movement. In these cases, new free barbed ends of actin ®laments are created, and actin subunits add to those ends. In addition, the actin ®laments generally form a branching network, with the barbed ends oriented toward the membrane or object being moved. Arp2/3 complex is a key component of these processes; it forms the branch points and creates new actin ®laments with free barbed ends. www.current-opinion.com
Alone, Arp2/3 complex is inactive. In this review, we consider what proteins bind to and/or activate Arp2/3 complex, thus potentially targeting its actin nucleation activity in cells. We also consider the nature of the molecular mechanisms by which these proteins activate Arp2/3 complex. In particular, we address the question of how the effects of multiple activators are integrated by Arp2/3 complex. We consider results from in vitro and in vivo approaches. The Arp2/3 complex is a seven-protein complex containing two actin-related proteins (Arp2 and Arp3), and ®ve otherwise unique polypeptides (ARPC1±5) [1]. Arp2/3 complex binds to the side of a pre-existing actin ®lament and nucleates a new ®lament with a free barbed end; the mother and daughter ®laments form a stiff branch with a 708 angle between them. Arp2 and Arp3 appear to occupy the positions corresponding to the ®rst two subunits of the daughter ®lament [2], suggesting that nucleation occurs because Arp2 and Arp3 resemble the subunits at the barbed end of an actin ®lament. Activation of Arp2/3 complex probably involves a large conformational change, especially in the relative positions of Arp2 and Arp3 [1,2].
Arp2/3 complex interactors: the WASP and cortactin families
Two major protein families that bind and activate Arp2/3 complex have been studied extensively. The Wiskott± Aldrich syndrome protein (WASP) family has ®ve members ± WASP, neuronal WASP (N-WASP) and SCAR/ WAVE1±3 (suppressor of cAMP receptor/WASP family verprolin-homologous protein 1±3). The cortactin family includes cortactin and hematopoiesis-speci®c protein (HS1). Proteins of both families contain an acidic (A) domain, which binds Arp2/3 complex (Table 1). In addition, the CARMIL protein binds Arp2/3 complex, as well as capping protein and myosin-I. CARMIL was discovered and studied in amoeba, and homologues are present in a wide variety of multicellular eukaryote genomes. The A domain of CARMIL can weakly activate Arp2/3 complex [3]. WASP-family proteins are evolutionarily conserved, with representatives found from yeast to humans. Cortactin also appears to be highly conserved, with genes in mouse, human, chicken, Drosophila, Xenopus, sponge and sea urchin [4]. WASP-family members all contain a carboxy-terminal activation domain (WA domain), consisting of one or two WH2 (WASP homology 2) domains, which bind monomeric actin, and an A domain. N-WASP and WASP are autoinhibited, owing to the amino-terminal portion of the protein folding onto and thus masking Current Opinion in Cell Biology 2003, 15:23±30
24 Cell structure and dynamics
Table 1 A-domain or acidic region of Arp2/3 complex interactors. Protein
A domain alignment
Majority Human N-WASP Human WASP S. cerevisiae Las17p S. pombe Wsp1p Human SCAR1 Human SCAR2 Human SCAR3 D. discoideum SCAR S. cerevisiae Myo3p S. cerevisiae Myo5p S. pombe Myo1p A. nidulans MYO1A S. cerevisiae Abp1pN S. cerevisiae Abp1pC S. cerevisiae Pan1p S. cerevisiae Dos2p Human KIF4a Human cortactin Human HS-1 D. discoideum CARMILb
±A±L±R- - - - ± - - - ± - - - -DEDDDDDW± MEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED MHVMQKRSRAIHSSDEGEDQAG±DEDEDDEWDD ALAAALNKRKTKV- - - - - - -GAHDDMDNGDDW ALASALNQRKTKV- - - - ±AQSDEEDEDDDEWD DVATILSRRIA-VEYSDS- - -EDDSEFDEVDWLE DVATILSRRIA-VEYSDS±EDDSSEFDEDDWSD DVATILSRRIA-VEYSDS- - -DDDSEFDENDWSD NVADILARRIA-WAG-DS±DSSEDESDDSDWD GLASALAARANKMRAESADDDDNDDGDDDDDW GLANALAARANKMRLESDDEEAN-EDEEEDDW SLADALRMRASAVRGS- - - - ± - - - -DEEEDW GLAEALRRRQSAMQGKQ- - - - - - ÐDDDDDW TPSPAPAAKIS-SRVNDNN- - - - - - ±DDDDWNE TDYKKIGNPLPGMHI±EADNEEEPEENDDDWDDDEDE ARAAEMQRRIQRGL- - - - ± - - - -DEDEDDGWSD VSQEGLKDVSDHVGLANKDESK±DDDDDDDWE MDIEDLKYCSEHSVNEHEDGDGDDDEGDDEEWKP MWKASAGHAVSIAQ- - - - - - -DDAGADDWET MWKSVVGHDVSVSV- - - - - - ±ETQGDDWDT DVDEQQIHSTLVKGAGAELSSRAHECFISALDIASDYTYEKITIGLDS VFKDLILEESQAQNEASGATPIPD ESADTRAES-LPETSTAD- - - - ± - - - - - ±DTPSEASEEEPNLGSES SDTSDLPTTTTDAAAAAEEAE
A. castellanii CARMILb
Net charge
Locationc (aa number)
16 10 01 07 09 10 09 08 09 09 03 02 04 15 04 10 14 04 04 10
C (472±505) C (471±502) C (609±633) C (548±574) C (530±559) C (468±498) C (473±502) C (415±443) C (1240±1271) C (1189±1219) C (1196±1217) C (1227±1249) I (181±205) I (410±445) I (1259±1480) C (280±310) I (1039±1232) N (1±24) N (1±23) I (792±863)
16
I (871±925)
a
Human chromokinesin protein KIF4 contains a conserved tail region with A-domain-like sequence. A GST fusion protein of human KIF4 tail (amino acids [aa] 1001±1232) did not enhance Arp2/3-mediated actin nucleation (W-L Lee, TD Pollard and JA Cooper, unpublished data). b The acidic regions of CARMIL proteins [3] were only aligned against each other. c The location of each A domain or acidic region in the primary sequence: N, amino terminus; C, carboxyl terminus; I, internal. Highly conserved residues are printed in bold typeface. Acidic residues are underlined.
the carboxy-terminal portion. The autoinhibition is relieved when signaling molecules, such as phosphatidylinositol 4,5-bisphosphate, activated Rho-family GTPases, or tyrosine kinase adaptor molecules bind to the basic, GBD, and proline-rich domains, respectively, located in the amino-terminal portion of WASP [5]. Unlike N-WASP and WASP, SCAR/WAVE proteins are not autoinhibited [6]. Puri®ed recombinant WAVE proteins are fully active, but native WAVE1 puri®ed from brain appears to be inhibited by the binding of additional proteins. In these studies, activation could be achieved by the binding of activated Rac1 or Nck, which dissociated some of the WAVE1-binding partners [7]. The actin monomer bound to the WH2 domain appears to add to the Arp2/3 complex and thus promote nucleation of the new daughter ®lament [5]. A proline-rich domain amino-terminal to the WH2 domain binds pro®lin, which binds monomeric actin and also promotes nucleation [8]. Like WASP-family proteins, cortactin has an acidic domain that binds Arp2/3 complex. In contrast to WASPs, the A domain of cortactin is near its amino terminus [4]. Cortactin also has a central tandem repeat domain that binds to actin ®laments. The A domain of cortactin alone is not suf®cient to activate Arp2/3 complex. The activation induced by cortactin also requires actin ®lament Current Opinion in Cell Biology 2003, 15:23±30
binding by cortactin and may be caused by cortactin bringing Arp2/3 complex into proximity with the actin ®lament [9,10], which is an important co-factor for activation [6]. Cortactin also contains a Src homology 3 (SH3) domain at its carboxyl terminus, which binds several proteins, including dynamin, FGD1, Shank, WASP-interacting protein (WIP) and ZO-1 ([4]; P Hou et al., and A Kinley et al., unpublished data). The effect of SH3-domain-binding proteins appears to differ depending on the protein. For example, WIP binding strongly enhances actin nucleation induced by cortactin and Arp2/3 complex, presumably because WIP binds actin monomers (A Kinley et al., unpublished data). WIP does not enhance activation of Arp2/3 complex by N-WASP, perhaps because N-WASP already binds monomeric actin [11]. On the other hand, only weak activation of cortactin and Arp2/3 complex is achieved by dynamin or the SH3-binding region of FGD1, which seems to involve multimerization of cortactin ([12]; P Hou et al., unpublished data). A monomeric form of the SH3-binding domain of FGD1 has no effect on activation of Arp2/3 complex by cortactin (P Hou et al., unpublished data), con®rming that cortactin is not autoinhibited [9,10]. Thus, SH3 ligands may target and/or activate cortactin in vivo. Cortactin was discovered as a prominent substrate for tyrosine phosphorylation by Src kinase [4]. The three www.current-opinion.com
Integration of signals to the Arp2/3 complex Weaver et al. 25
tyrosine residues phosphorylated by Src are located between the central F-actin binding domain and the SH3 domain. The effect of tyrosine phosphorylation on cortactin activity is unclear. In vitro, removal of the carboxy-terminal portion, including the Src phosphorylation sites, did not affect the ability of cortactin to activate the Arp2/3 complex [9,10]. In vivo, expression of a cortactin mutated in all three Src tyrosine phosphorylation sites decreased the rate of endothelial cell migration in culture by a small amount [13] and decreased the number of breast cancer cell metastases to bone in nude mice [14]. The mechanism of these in vivo effects remains to be investigated. Compared with N-WASP, cortactin is a weak activator of the Arp2/3 complex. The difference may be explained by the fact that N-WASP, but not cortactin, binds monomeric actin, which may then bind to the Arp2/3 complex during nucleation. Alternatively, the A domains of cortactin and N-WASP bind to Arp2/3 complex in different ways, so the interactions unique to N-WASP may be more effective at inducing the active conformation of Arp2/3 complex. Cortactin can be chemically crosslinked to only a single subunit, Arp3, whereas N-WASP can be crosslinked to Arp2, Arp3 and ARPC1/p40 [15]. As noted above, the ability of cortactin to bind F-actin is also necessary for it to activate the Arp2/3 complex, so the activation of the Arp2/3 complex may be caused by the increased probability of association with F-actin. The fact that cortactin binds simultaneously to the Arp2/3 complex and F-actin lends cortactin an additional activity, which is to stabilise and prolong the lifetime of Arp2/3 complexinduced branches [9]. In vertebrate tissues, cortactin is expressed in polarised epithelial cells and some muscle cells [16]. It also is expressed in a variety of cultured cells, including ®broblasts, carcinoma cell lines, and neuronal cell lines [4]. HS-1 is a cortactin family member expressed in haematopoietic cells; the domain structure of HS-1 is similar to that of cortactin [4]. WASP, cortactin and other Arp2/3 complex interactors in yeast
Budding and ®ssion yeast contain one WASP-family protein, Las17p and Wsp1p, respectively (Table 2). Like mammalian WASPs, the yeast proteins contain a WH1 domain, polyproline regions, a WH2 domain, and an A domain. However, they lack a GBD sequence, and they apparently do not bind Rho-family GTPases. The A domain of Las17p is necessary and suf®cient to bind Arp2/3 complex [17,18]. Sequence analysis in yeast shows no cortactin homologue, but Abp1p of Saccharomyces cerevisiae (ScAbp1p) may function similarly to cortactin. ScAbp1p contains an F-actinbinding ADF (actin depolymerizing factor) homology www.current-opinion.com
domain, two A domains, and a carboxy-terminal SH3 domain that mediates interactions with adaptor proteins. Puri®ed ScAbp1p binds Arp2/3 complex stoichiometrically and can form a ternary complex with Arp2/3 complex and actin ®laments [19]. ScAbp1p associated with Arp3, but not other ARP complex components (ARPCs), in a recent systematic co-precipitation study [20]. Mammalian and ®ssion yeast Abp1 homologues exist [21±23] but may not interact directly with the Arp2/3 complex, because they lack identi®able A domains. The F-actin-binding protein coronin is conserved from yeast to mammals, but its function has been elusive. Recent work in yeast now shows that the coiled-coil domain of coronin (Sc Crn1p) interacts with puri®ed Arp2/3 complex and can inhibit Arp2/3-complex-mediated actin nucleation [24]. Type-I myosins in yeasts and fungi, but not higher organisms, contain a carboxy-terminal A domain that binds the Arp2/3 complex [25,26,27]. Budding yeast myosin-I proteins (Myo3p and Myo5p) bind two other proteins that also interact with Arp2/3 complex. First, their SH3 domains bind Las17p [26,27,28]. Second, their IQ (light chain binding) motifs bind calmodulin [29], which interacts with the Arp2/3 complex component Arc35p/ARPC2 in two-hybrid assays and by immunoprecipitation from cell extracts [30]. Finally, Pan1p of S. cerevisiae, a putative Eps15 homologue, binds puri®ed Arp2/3 complex and contains an A domain. This A domain may or may not be conserved in other yeast or mammals [31]. Yeast components that activate Arp2/3 complex
In S. cerevisiae Las17p, Abp1p, and Pan1p have been shown to activate the Arp2/3 complex [18,19,31]. We found that the carboxyl terminus of another A-domaincontaining protein, Dos2p, was also capable of activating the Arp2/3 complex; however, a dos2D mutant had no phenotype associated with actin function (ME Young, W-L Lee and JA Cooper, unpublished data). Homologues of these four proteins exist in ®ssion yeast, but enhanced Arp2/3 complex activity has only been demonstrated for the WASP homologue Wsp1p (V Sirotkin and TD Pollard, personal communication). In addition, in ®ssion yeast the tail of Myo1p, the myosin-I in this organism, activates the Arp2/3 complex [25]. Both the A domain and the TH2 (tail homology 2) domain appeared to be required for activity, and the potency of Myo1p was further increased by the addition of ®ssion yeast verprolin/WIP (V Sirotkin and TD Pollard, personal communication). The A domain of a budding yeast myosin-I, Myo3p, was not suf®cient to activate Arp2/3 complex, but it did have activity when arti®cially coupled to the WH2 actin-monomer-binding domain of budding yeast verprolin/WIP(Vrp1p) [32]. Current Opinion in Cell Biology 2003, 15:23±30
26 Cell structure and dynamics
Table 2 Budding and fission yeast Arp2/3 complex interactors: constructs and assays used. Protein
Construct
Binds Arp2/3 complexa
Activates Arp2/3 complexb
Binds actinc
References
Las17p
FL
(IP)
G
FL±DA WA
(IP) ND
ND (
