Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.     

The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex.

Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization.     

Identification of another actin-related protein (Arp) 2/3 complex binding site in neural Wiskott-Aldrich syndrome protein (N-WASP) that complements actin polymerization induced by the Arp2/3 complex activating (VCA) domain of N-WASP.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.     

Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex

The Wiskott-Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a Kd of 0.6 microM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable DeltaH of -10 kcal mol(-1) drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 microM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott-Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 complex.     

A neural Wiskott-Aldrich Syndrome protein-mediated pathway for localized activation of actin polymerization that is regulated by cortactin

Activation of the epidermal growth factor (EGF) receptor can stimulate actin polymerization via the Arp2/3 complex using a number of signaling pathways, and specific stimulation conditions may control which pathways are activated. We have previously shown that localized stimulation of EGF receptor with EGF bound to beads results in localized actin polymerization and protrusion. Here we show that the actin polymerization is dependent upon activation of the Arp2/3 complex by neural Wiskott-Aldrich Syndrome protein (N-WASP) via Grb2 and Nck2. Suppression of Grb2 or Nck2 results in loss of localization of N-WASP at the activation site and reduced actin polymerization. Although cortactin has been found to synergize with N-WASP for Arp2/3-dependent actin polymerization in vitro, we find that cortactin can restrict N-WASP localization around EGF-bead-induced protrusions. In addition, cortactin-deficient cells have increased lamellipod dynamics but show reduced net translocation, suggesting that cortactin can contribute to cell polarity by controlling the extent of Arp2/3 activation by WASP family members and the stability of the F-actin network.     

Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.     

Erk/Src phosphorylation of cortactin acts as a switch on-switch off mechanism that controls its ability to activate N-WASP

The Arp2/3 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP family members and by the acidic N-terminal and F-actin-binding repeat region of cortactin, which possesses a C-terminal SH3 domain. Cortactin is a target for phosphorylation by Src tyrosine kinases and by serine/threonine kinases that include Erk. Here we demonstrate that cortactin binds N-WASP and WASP via its SH3 domain, induces in vitro N-WASP-mediated actin polymerization, and colocalizes with N-WASP and WASP at sites of active actin polymerization. Erk phosphorylation and a mimicking S405,418D double mutation enhanced cortactin binding and activation of N-WASP. In contrast, Src phosphorylation inhibited the ability of cortactin previously phosphorylated by Erk, and that of S405,418D double mutant cortactin, to bind and activate N-WASP. Furthermore, Y-->D mutation of three tyrosine residues targeted by Src (Y421, Y466, and Y482) inhibited the ability of S405,418D cortactin to activate N-WASP. We propose that Erk phosphorylation liberates the SH3 domain of cortactin from intramolecular interactions with proline-rich regions, causing it to synergize with WASP and N-WASP in activating the Arp2/3 complex, and that Src phosphorylation terminates cortactin activation of N-WASP and WASP.     

SCAR knockouts in Dictyostelium: WASP assumes SCAR's position and upstream regulators in pseudopods

Under normal conditions, the Arp2/3 complex activator SCAR/WAVE controls actin polymerization in pseudopods, whereas Wiskott-Aldrich syndrome protein (WASP) assembles actin at clathrin-coated pits. We show that, unexpectedly, Dictyostelium discoideum SCAR knockouts could still spread, migrate, and chemotax using pseudopods driven by the Arp2/3 complex. In the absence of SCAR, some WASP relocated from the coated pits to the leading edge, where it behaved with similar dynamics to normal SCAR, forming split pseudopods and traveling waves. Pseudopods colocalized with active Rac, whether driven by WASP or SCAR, though Rac was activated to a higher level in SCAR mutants. Members of the SCAR regulatory complex, in particular PIR121, were not required for WASP regulation. We thus show that WASP is able to respond to all core upstream signals and that regulators coupled through the other members of SCAR's regulatory complex are not essential for pseudopod formation. We conclude that WASP and SCAR can regulate pseudopod actin using similar mechanisms.     

Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities

Background: Assembly and organization of actin filaments are required for many cellular processes, including locomotion and division. In many cases, actin assembly is initiated when proteins of the WASP/Scar family respond to signals from Rho family G proteins and stimulate the actin-nucleating activity of the Arp2/3 complex. Two questions of fundamental importance raised in the study of actin dynamics concern the molecular mechanism of Arp2/3-dependent actin nucleation and how different signaling pathways that activate the same Arp2/3 complex produce actin networks with different three-dimensional architectures?Results: We directly compared the activity of the Arp2/3 complex in the presence of saturating concentrations of the minimal Arp2/3-activating domains of WASP, N-WASP, and Scar1 and found that each induces unique kinetics of actin assembly. In cell extracts, N-WASP induces rapid actin polymerization, while Scar1 fails to induce detectable polymerization. Using purified proteins, Scar1 induces the slowest rate of nucleation. WASP activity is 16-fold higher, and N-WASP activity is 70-fold higher. The data for all activators fit a mathematical model in which one activated Arp2/3 complex, one actin monomer, and an actin filament combine into a preactivation complex which then undergoes a first-order activation step to become a nucleus. The differences between Scar and N-WASP activity are explained by differences in the rate constants for the activation step. Changing the number of actin binding sites on a WASP family protein, either by removing a WH2 domain from N-WASP or by adding WH2 domains to Scar1, has no significant effect on nucleation activity. The addition of a three amino acid insertion found in the C-terminal acidic domains of WASP and N-WASP, however, increases the activity of Scar1 by more than 20-fold. Using chemical crosslinking assays, we determined that both N-WASP and Scar1 induce a conformational change in the Arp2/3 complex but crosslink with different efficiencies to the small molecular weight subunits p18 and p14.Conclusion: The WA domains of N-WASP, WASP, and Scar1 bind actin and Arp2/3 with nearly identical affinities but stimulate rates of actin nucleation that vary by almost 100-fold. The differences in nucleation rate are caused by differences in the number of acidic amino acids at the C terminus, so each protein is tuned to produce a different rate of actin filament formation. Arp2/3, therefore, is not regulated by a simple on-off switch. Precise tuning of the filament formation rate may help determine the architecture of actin networks produced by different nucleation-promoting factors.     

Membrane-targeted WAVE mediates photoreceptor axon targeting in the absence of the WAVE complex in Drosophila

A tight spatial-temporal coordination of F-actin dynamics is crucial for a large variety of cellular processes that shape cells. The Abelson interactor (Abi) has a conserved role in Arp2/3-dependent actin polymerization, regulating Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE). In this paper, we report that Abi exerts nonautonomous control of photoreceptor axon targeting in the Drosophila visual system through WAVE. In abi mutants, WAVE is unstable but restored by reexpression of Abi, confirming that Abi controls the integrity of the WAVE complex in vivo. Remarkably, expression of a membrane-tethered WAVE protein rescues the axonal projection defects of abi mutants in the absence of the other subunits of the WAVE complex, whereas cytoplasmic WAVE only slightly affects the abi mutant phenotype. Thus complex formation not only stabilizes WAVE, but also provides further membrane-recruiting signals, resulting in an activation of WAVE.     

Phosphorylation of the WASP-VCA domain increases its affinity for the Arp2/3 complex and enhances actin polymerization by WASP

Wiskott-Aldrich syndrome protein (WASP) and neural (N)-WASP regulate dynamic actin structures through the ability of their VCA domains to bind to and stimulate the actin nucleating activity of the Arp2/3 complex. Here we identify two phosphorylation sites in the VCA domain of WASP at serines 483 and 484. S483 and S484 are substrates for casein kinase 2 in vitro and in vivo. Phosphorylation of these residues increases the affinity of the VCA domain for the Arp2/3 complex 7-fold and is required for efficient in vitro actin polymerization by the full-length WASP molecule. We propose that constitutive VCA domain phosphorylation is required for optimal stimulation of the Arp2/3 complex by WASP.     

Critical conformational changes in the Arp2/3 complex are induced by nucleotide and nucleation promoting factor

Actin nucleation and branching by the Arp2/3 complex is tightly regulated by activating factors. However, the mechanism of Arp2/3 complex activation remains unclear. We used fluorescence resonance energy transfer (FRET) to probe the conformational dynamics of the Arp2/3 complex accompanying its activation. We demonstrate that nucleotide binding promotes a substantial conformational change in the complex, with distinct conformations depending on the bound nucleotide. Nucleotide binding to each Arp is critical for activity and is coupled to nucleation promoting factor (NPF) binding. The binding of Wiskott-Aldrich syndrome protein (WASP) family NPFs induces further conformational reorganization of the Arp2/3 complex, and the ability to promote this conformational reorganization correlates with activation efficiency. Using an Arp2/3 complex that is fused to the actin binding domain of WASP, we confirm that the NPF-induced conformational change is critical for activation, and that the actin and Arp2/3 binding activities of WASP are separable, but are independently essential for activity.     

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

Scar1 and the related Wiskott–Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex

Background: The actin-related proteins Arp2 and Arp3 are part of a seven-protein complex which is localized in the lamellipodia of a variety of cell types, and in actin-rich spots of unknown function. The Arp2/3 complex enhances actin nucleation and causes branching and crosslinking of actin filaments in vitro; in vivo it is thought to drive the formation of lamellipodia and to be a control center for actin-based motility. The Wiskott–Aldrich syndrome protein, WASP, is an adaptor protein implicated in the transmission of signals from tyrosine kinase receptors and small GTPases to the actin cytoskeleton. Scar1 is a member of a new family of proteins related to WASP, and it may also have a role in regulating the actin cytoskeleton. Scar1 is the human homologue of Dictyostelium Scar1, which is thought to connect G-protein-coupled receptors to the actin cytoskeleton. The mammalian Scar family contains at least four members. We have examined the relationships between WASP, Scar1, and the Arp2/3 complex.Results: We have identified WASP and its relative Scar1 as proteins that interact with the Arp2/3 complex. We have used deletion analysis to show that both WASP and Scar1 interact with the p21 subunit of the Arp2/3 complex through their carboxyl termini. Overexpression of carboxy-terminal fragments of Scar1 or WASP in cells caused a disruption in the localization of the Arp2/3 complex and, concomitantly, induced a complete loss of lamellipodia and actin spots. The induction of lamellipodia by platelet-derived growth factor was also suppressed by overexpression of the fragment of Scar1 that binds to the Arp2/3 complex.Conclusions: We have identified a conserved sequence domain in proteins of the WASP family that binds to the Arp2/3 complex. Overexpression of this domain in cells disrupts the localization of the Arp2/3 complex and inhibits lamellipodia formation. Our data suggest that WASP-related proteins may regulate the actin cytoskeleton through the Arp2/3 complex.     

Acceleration of yeast actin polymerization by yeast Arp2/3 complex does not require an Arp2/3-activating protein

The Arp2/3 complex creates filament branches leading to an enhancement in the rate of actin polymerization. Work with Arp complexes from different sources indicated that it was inactive by itself, required an activating factor such as the Wiskott-Aldrich syndrome protein (WASP), and might exhibit a preference for ATP or ADP-P(i) actin. However, with yeast actin, P(i) release is almost concurrent with polymerization, eliminating the presence of an ADP-P(i) cap. We thus investigated the ability of the yeast Arp2/3 complex (yArp2/3) to facilitate yeast actin polymerization in the presence and absence of the Arp2/3-activating factor Las17p WA. yArp2/3 significantly accelerates yeast actin but not muscle actin polymerization in the absence of Las17p WA. The addition of Las17p WA further enhances yeast actin polymerization by yArp2/3 and allows the complex to now assist muscle actin polymerization. This actin isoform difference is not observed with bovine Arp2/3 complex, because the neural WASP VCA fragment is required for polymerization of both actins. Observation of individual branching filaments showed that Las17p WA increased the persistence of filament branches. Compared with wild type actin, the V159N mutant actin, proposed to be more ATP-like in behavior, exhibited an enhanced rate of polymerization in the presence of the yArp2/3 complex. yArp2/3 caused a significant rate of P(i) release prior to observation of an increase in filament mass but while branched structures were present. Thus, yeast F-actin can serve as a primary yArp2/3-activating factor, indicating that a newly formed yeast actin filament has a topology, unlike that of muscle actin, that is recognized specifically by yArp2/3.     

Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer-binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell-cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.     

Contingent phosphorylation/dephosphorylation provides a mechanism of molecular memory in WASP

Cells can retain information about previous stimuli to produce distinct future responses. The biochemical mechanisms by which this is achieved are not well understood. The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho-family GTPase Cdc42, whose activation leads to stimulation of the actin nucleating assembly, Arp2/3 complex. We demonstrate that efficient phosphorylation and dephosphorylation of WASP at Y291 are both contingent on binding to activated Cdc42. Y291 phosphorylation increases the basal activity of WASP toward Arp2/3 complex and enables WASP activation by new stimuli, SH2 domains of Src-family kinases. The requirement for contingency in both phosphorylation and dephosphorylation enables long-term storage of information by WASP following decay of GTPase signals. This biochemical circuitry allows WASP to respond to the levels and timing of GTPase and kinase signals. It provides mechanisms to specifically achieve transient or persistent actin remodeling, as well as long-lasting potentiation of actin-based responses to kinases.