Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex.
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The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament-a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex. (+info)
The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.
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Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton. (+info)
Phosphorylation of WAVE downstream of mitogen-activated protein kinase signaling.
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WAVE is a Wiskott-Aldrich syndrome protein (WASP)-family protein that functions in membrane-ruffling formation induced by Rac, a Rho family small GTPase. Here we report that WAVE is a phosphoprotein whose phosphorylation increases in response to various external stimuli that activate mitogen-activated protein (MAP) kinase signaling. When Swiss 3T3 cells are stimulated with platelet-derived growth factor, electrophoretic mobility shift occurs to WAVE, which reflects hyperphosphorylation. This is perfectly inhibited by the addition of PD98059, a specific inhibitor of MAP kinase kinase. Indeed, the ectopic expression of an activated mutant of MAP kinase kinase induces WAVE mobility shift. When MAP kinase activation is suppressed by PD98059, the intensity of platelet-derived growth factor-induced membrane ruffling is greatly reduced. In various cancer cell lines, the amount of WAVE mobility shift was found to increase significantly, suggesting the importance of WAVE hyperphosphorylation in the formation of membrane ruffles and oncogenic transformation. (+info)
Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold.
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WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl. (+info)
Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex.
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ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3. (+info)
Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP.
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All WASP family proteins share a common C terminus that consists of the verprolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which they activate Arp2/3 complex-induced actin polymerization. In this study, we characterized the Arp2/3 complex-mediated actin polymerization activity of VCA fragments of all of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3. All of the VCA fragments stimulated the nucleating activity of Arp2/3 complex. Among them, N-WASP VCA, which possesses two tandem V motifs, had a more potent activity than other VCA proteins. The chimeric protein experiments revealed that the V motif was more important to the activation potency than the CA region; two V motifs were required for full activity of N-WASP. COS7 cells overexpressing N-WASP form microspikes in response to epidermal growth factor. However, when a chimeric protein in which the VCA region of N-WASP is replaced with WAVE1 VCA was overexpressed, microspike formation was suppressed. Interestingly, when the N-WASP VCA region was replaced with WAVE1 VCA, having two V motifs, this chimeric protein could induce microspike formation. These results indicate that strong activation of Arp2/3 complex by N-WASP is mainly caused by its two tandem V motifs, which are essential for actin microspike formation. (+info)
Scar/WAVE is localised at the tips of protruding lamellipodia in living cells.
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Cell motility entails the extension of cytoplasmic processes, termed lamellipodia and filopodia. Extension is driven by actin polymerisation at the tips of these processes via molecular complexes that remain to be characterised. We show here that a green fluorescent protein (GFP) fusion of the Wiskott-Aldrich syndrome protein family member Scar1/WAVE1 is specifically recruited to the tips of lamellipodia in living B16F1 melanoma cells. Scar1-GFP was recruited only to protruding lamellipodia and was absent from filopodia. The localisation of Scar was facilitated by the finding that the formerly described inhibition of lamellipodia formation by ectopical expression of Scar, could be overcome by the treatment of cells with aluminium fluoride. These findings show that Scar is strategically located at sites of actin polymerisation specifically engaged in the protrusion of lamellipodia. (+info)
N-WASP, WAVE and Mena play different roles in the organization of actin cytoskeleton in lamellipodia.
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WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. We observed the localization of the WASP family proteins, N-WASP and WAVE, and the Ena/VASP family protein, Mena, in protruding lamellipodia. Rat fibroblast cell line 3Y1 protruded lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 were concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena were observed to localize at the lamellipodium edge. Interestingly, Mena tended to concentrate at the microspike tips but WAVE did not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, was much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, was localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE was distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42. (+info)