Wiscott-Aldrich Syndrome Protein Research Articles

Under Pathogen Attack

Under Pathogen Attack

Regulation of Actin Ring Formation by Rho GTPases in Osteoclasts

Actin ring formation is a prerequisite for osteoclast bone resorption. Although gelsolin null osteoclasts failed to exhibit podosomes, actin ring was observed in these osteoclasts. Wiscott-Aldrich syndrome protein (WASP) was observed in the actin ring of gelsolin null osteoclast. Osteoclasts stimulated with osteopontin simulated the effects of Rho and Cdc42 in phosphatidylinositol 4,5-bisphosphate (PIP2) association with WASP as well as formation of podosomes, peripheral microfilopodia-like structures, and actin ring. To explore the potential functions of Rho and Cdc42, TAT-mediated delivery of Rho proteins into osteoclasts was performed. Although Rho and Cdc42 are required for actin ring formation, transduction of either one of the proteins alone is insufficient for this process. Addition of osteopontin to osteoclasts transduced with Cdc42Val12 or transduction of osteoclasts with both RhoVal14 and Cdc42Val12 augments the formation of WASP-Arp2/3 complex and actin ring. Neomycin, an antibiotic, blocked the effects of osteopontin or TAT-RhoVal14 on PIP2 interaction with WASP. WASP distribution was found to be cytosolic in these osteoclasts. Depletion of WASP by short interfering RNA-mediated gene silencing blocked actin polymerization as well as actin ring formation in osteoclasts. These results suggest that Rho-mediated PIP2 interaction with WASP may contribute to the activation and membrane targeting of WASP. Subsequent interaction of Cdc42 and Arp2/3 with WASP may enhance cortical actin polymerization in the process of actin ring formation in osteoclasts.

Selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice

During a productive infection, the prototype strain of parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations to the fibroblast host cell A9, resulting in cell lysis and progeny virus release. In order to understand the mechanisms underlying these changes, we characterized the fate of various cytoskeletal filaments and investigated the nuclear/cytoplasmic compartmentalization of infected cells. While most pronounced effects could be seen on micro- and intermediate filaments, manifest in dramatic rearrangements and degradation of filamentous (F-)actin and vimentin structures, only little impact could be seen on microtubules or the nuclear envelope during the entire monitored time of infection. To further analyze the disruption of the cytoskeletal structures, we investigated the viral impact on selective regulatory pathways. Thereby, we found a correlation between microtubule stability and MVM-induced phosphorylation of α/β tubulin. In contrast, disassembly of actin filaments late in infection could be traced back to the disregulation of two F-actin associated proteins gelsolin and Wiscott–Aldrich Syndrome Protein (WASP). Thereby, an increase in the amount of gelsolin, an F-actin severing protein was observed during infection, accounting for the disruption of stress fibers upon infection. Concomitantly, the actin polymerization activity also diminished due to a loss of WASP, the activator protein of the actin polymerization machinery the Arp2/3 complex. No effects could be seen in amount and distribution of other F-actin regulatory factors such as cortactin, cofilin, and profilin. In summary, the selective attack of MVM towards distinct host cell cytoskeletal structures argues for a regulatory feature during infection, rather than a collapse of the host cell as a mere side effect of virus production.

Inhibiting the Arp2/3 complex limits infection of both intracellular mature vaccinia virus and primate lentiviruses.

Characterizing cellular factors involved in the life cycle of human immunodeficiency virus type 1 (HIV-1) is an initial step toward controlling replication of HIV-1. Actin polymerization mediated by the Arp2/3 complex has been found to play a critical role in some pathogens' intracellular motility. We have asked whether this complex also contributes to the viral life cycles including that of HIV-1. We have used both the acidic domains from actin-related protein (Arp) 2/3 complex-binding proteins such as the Wiscott-Aldrich syndrome protein (N-WASP) or cortactin, and siRNA directing toward Arp2 to inhibit viral infection. HIV-1, simian immunodeficiency virus (SIV), and intracellular mature vaccinia virus (IMV) were sensitive to inhibition of the Arp2/3 complex, whereas MLV, HSV-1, and adenovirus were not. Interestingly, pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) overcame this inhibition. Constitutive inhibition of the Arp2/3 complex in the T-cell line H9 also blocked replication of HIV-1. These data suggested the existence of an Arp2/3 complex-dependent event during the early phase of the life cycles of both primate lentiviruses and IMV. Inhibiting the HIV-1's ability to activate Arp2/3 complex could be a potential chemotherapeutic intervention for acquired immunodeficiency syndrome (AIDS).

Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.

Regulation of SPIN90 Phosphorylation and Interaction with Nck by ERK and Cell Adhesion

SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation. We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, betaPIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins. It appears that betaPIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix. The betaPIX.WASP.SPIN90.Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes. The SPIN90.betaPIX.WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90.Nck and SPIN90.betaPIX interactions, resulting in detachment of cells from extracellular matrix. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90.betaPIX.WASP complex and Nck. It thus appears that the interaction of the betaPIX.WASP.SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation.

Sequential Interaction of Actin-related Proteins 2 and 3 (Arp2/3) Complex with Neural Wiscott-Aldrich Syndrome Protein (N-WASP) and Cortactin during Branched Actin Filament Network Formation

The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells. Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization. In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished. Analysis of the cells infected with Shigella demonstrated that N-WASP located at the tip of the bacterium, whereas cortactin accumulated in the comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquired 20 nm affinity for the Arp2/3 complex during actin polymerization. The interaction of VCA with the Arp2/3 complex was reduced in the presence of both cortactin and actin. Moreover, VCA reduced its affinity for Arp2/3 complex at branching sites that were stabilized by phalloidin. These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex.

Fast and Slow Actin Nucleation by WASP Proteins

Members of the Wiscott-Aldrich syndrome protein (WASP) and the Scar family of actin binding proteins stimulate actin filament formation in response to signals from the Rho family of guanosine triphosphatases. Zalevsky et al. measured actin nucleation in vitro of Acanthamoeba extracts in the presence of peptides containing the actin-binding domains (WH2) and the acidic residues of the COOH-terminals of three members of the family: N-WASP, Scar1, and WASP. N-WASP with two WH2 domains was able to induce actin nucleation with the fastest kinetics, whereas Scar1 was the slowest at inducing actin nucleation. Analysis of chimeric proteins showed that the difference was not due to the presence of two WH2 domains, but rather was attributed to differences in the acidic domain. The N-WASP peptide induced the highest number of branches in the actin filaments. Although both N-WASP and Scar1 induced conformational changes in the actin-nucleating protein Arp2/3; they appear to mediate their effects through interactions with different subunits of the Arp2/3 complex as seen in chemical crosslinking experiments. Thus, members of the same family can stimulate actin nucleation through different molecular mechanisms, allowing differences in cellular response to stimuli that promote actin filament formation. Higgs discusses the various models for actin nucleation. J. Zalevsky, L. Lempert, H. Kranitz, R. D. Mullins, Different WASP family proteins stimulate different Apr2/3 complex-dependent actin-nucleating activities. Curr. Biol. 11 , 1903-1913 (2001). [Online Journal] H. N. Higgs, Actin nucleation: Nucleation-promoting factors are not all equal. Curr. Biol. 11 , R1009-R1012 (2001). [Online Journal]

Fast and Slow Actin Nucleation by WASP Proteins

Members of the Wiscott-Aldrich syndrome protein (WASP) and the Scar family of actin binding proteins stimulate actin filament formation in response to signals from the Rho family of guanosine triphosphatases. Zalevsky et al. measured actin nucleation in vitro of Acanthamoeba extracts in the presence of peptides containing the actin-binding domains (WH2) and the acidic residues of the COOH-terminals of three members of the family: N-WASP, Scar1, and WASP. N-WASP with two WH2 domains was able to induce actin nucleation with the fastest kinetics, whereas Scar1 was the slowest at inducing actin nucleation. Analysis of chimeric proteins showed that the difference was not due to the presence of two WH2 domains, but rather was attributed to differences in the acidic domain. The N-WASP peptide induced the highest number of branches in the actin filaments. Although both N-WASP and Scar1 induced conformational changes in the actin-nucleating protein Arp2/3; they appear to mediate their effects through interactions with different subunits of the Arp2/3 complex as seen in chemical crosslinking experiments. Thus, members of the same family can stimulate actin nucleation through different molecular mechanisms, allowing differences in cellular response to stimuli that promote actin filament formation. Higgs discusses the various models for actin nucleation.J. Zalevsky, L. Lempert, H. Kranitz, R. D. Mullins, Different WASP family proteins stimulate different Apr2/3 complex-dependent actin-nucleating activities. Curr. Biol. 11, 1903-1913 (2001). [Online Journal]H. N. Higgs, Actin nucleation: Nucleation-promoting factors are not all equal. Curr. Biol. 11, R1009-R1012 (2001). [Online Journal]

Characterization of human palladin, a microfilament-associated protein.

Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, alpha-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the alpha-helical domain of ezrin and by Ig-domains 2-3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.

Effect of overexpression of LAS17 on stress tolerance and the stability of extrachromosomal DNA in Saccharomyces cerevisiae

The effects of the overexpression of LAS17/BEE1, which encodes a yeast protein exhibiting sequence homology to the Wiscott-Aldrich syndrome protein, on the cell growth of Saccharomyces cerevisiae were examined. Sake yeast strain UT-1 grows at a faster rate as a result of the overexpression of LAS17 than control cultures under various stresses such as high temperature, high ethanol concentration, and oxidative stress, and the tolerance to these stresses was increased compared with the control. Moreover, a high cell survival rate was attained with overexpression of LAS17, when cells in the stationary phase of the growth cycle were subjected to heat killing (48°C) or ethanol killing (20% v/v). In addition, the rate of induction of rho − was markedly reduced by overexpression of LAS17 when serine, tyrosine, and aspartic acid were used as N sources and the yeast was cultured at 35°C, while rho − strains in control cultures were induced at a high frequency. After the incubation of cells harboring a multicopy vector in YPD or synthetic complete medium, almost all of the cells inherited the vector at about 15 copies per cell as a result of the overexpression of LAS17, whereas the cells harboring the control vector accounted for only 15% of the total number of cells. These results suggest that Las17p might be a multifunctional protein involved in cell growth regulation, extrachromosomal DNA transportation and stress responses.

Effect of Overexpression of LAS17 on Stress Tolerance and the Stability of Extrachromosomal DNA in Saccharomyces cerevisiae.

The effects of the overexpression of LAS17/BEE1, which encodes a yeast protein exhibiting sequence homology to the Wiscott-Aldrich syndrome protein, on the cell growth of Saccharomyces cerevisiae were examined. Sake yeast strain UT-1 grows at a faster rate as a result of the overexpression of LAS17 than control cultures under various stresses such as high temperature, high ethanol concentration, and oxidative stress, and the tolerance to these stresses was increased compared with the control. Moreover, a high cell survival rate was attained with overexpression of LAS17, when cells in the stationary phase of the growth cycle were subjected to heat killing (48 degrees C) or ethanol killing (20% v/v). In addition, the rate of induction of rho- was markedly reduced by overexpression of LAS17 when serine, tyrosine, and aspartic acid were used as N sources and the yeast was cultured at 35 degrees C, while rho- strains in control cultures were induced at a high frequency. After the incubation of cells harboring a multicopy vector in YPD or synthetic complete medium, almost all of the cells inherited the vector at about 15 copies per cell as a result of the overexpression of LAS17, whereas the cells harboring the control vector accounted for only 15% of the total number of cells. These results suggest that Las17p might be a multifunctional protein involved in cell growth regulation, extrachromosomal DNA transportation and stress responses.

Cbl-b Is a Negative Regulator of Receptor Clustering and Raft Aggregation in T Cells

Stimulation of T cells via the antigen and costimulatory receptors leads to the organization of a supramolecular activation cluster called the immune synapse. We report that loss of the molecular adaptor Cbl-b in T cells frees antigen receptor–triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimulation. Introduction of the cbl-b mutation into a vav1 −/− background relieved the functional defects of vav1 −/− T cells and caused spontaneous autoimmunity. Wiscott Aldrich Syndrome protein (WASP) was found to be essential for deregulated proliferation and membrane receptor reorganization of cbl-b mutant T cells. Antigen receptor–triggered Ca 2+ mobilization, cytokine production, and receptor clustering can be genetically uncoupled in cbl-b mutant T cells. Thus, Cbl-b functions as a negative regulator of receptor clustering and raft aggregation in T cells.

Integration with WASPs

Many signaling pathways control cell shape, and these pathways ultimately do so, at least in part, by regulating polymerization of actin. Members of the WASP (Wiskott-Aldrich syndrome protein) family appear to integrate such signals and are thus subject to a complex array of regulatory mechanisms. Activation of WASP through the Rho family GTPase Cdc42 and interaction with phosphatidylinositol 4,5-bisphosphate (PIP 2 ) stimulate the Arp2/3 complex, which in turn promotes nucleation of actin filaments. Papers by Higgs and Pollard and Rohatgi et al . characterizing native WASP from bovine thymus and in vitro-translated N-WASP (where N refers to neuronal, even though this family member is widely expressed) and the Commentary by Zigmond provide new details of this multifaceted regulation. The NH 2 -terminus of WASP appears to interact with the COOH-terminus and block binding to Arp2/3. Activation by Cdc42 and PIP 2 reduces the autoinhibitory binding and exposes the Arp2/3 binding region. Rohatgi et al . use a mutant N-WASP insensitive to PIP 2 but sensitive to Cdc42 to show that the critical function of PIP 2 may actually be to activate Cdc42. Higgs and Pollard provide evidence that clustering of PIP 2 and Cdc42 on a membrane surface is important for efficient activation. Still more is to come, as investigators factor in the association of WASP proteins with receptors, viral proteins, other cytoskeletal proteins (profilin), and so on. Higgs, H.N., and Pollard, T.D. (2000) Activation by Cdc42 and PIP 2 of Wiscott-Aldrich Syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex. J. Cell Biol. 150 : 1311-1320. [Abstract] [Full Text] Rohatgi, R., Ho, H.-y.H., and Kirschner, M.W. (2000) Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-biphosphate. J. Cell Biol. 150 : 1299-1309. [Abstract] [Full Text] Zigmond, S.H. (2000) How WASP regulates actin polymerization. J. Cell Biol. 150 : F117-F119. [Full Text]

The p150-Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization.

The Jun N-terminal kinase (JNK) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization [1-3]. A role of the Drosophila JNK homologue, Basket (DJNK/Bsk), in the regulation of cell shape changes and actin reorganization arises from its function in the process of dorsal closure [4-6]. One potential mechanism for induction of cytoskeletal changes by JNK is via transcriptional activation of the decapentaplegic gene (dpp, a member of the TGFbeta superfamily) [6]. A direct link between JNK signalling and actin organization has not yet been found, however. We have identified a novel DJNK-interacting protein, p150-Spir, that belongs to the Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2) family of proteins involved in actin reorganization [7] [8]. It is a multidomain protein with a cluster of four WH2 domains, a modified FYVE zinc-finger motif [9], and a DEJL motif, a docking site for JNK [10], at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalized with F-actin and its overexpression induced clustering of filamentous actin around the nucleus. When coexpressed with p150-Spir in NIH 3T3 cells, JNK translocated to and colocalizes with p150-Spir at discrete spots around the nucleus. Carboxy-terminal sequences of p150-Spir were phosphorylated by JNK both in vitro and in vivo. We conclude that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization.

Identification and characterization of small GTPase-associated kinases.

AbstractRho-family signal-transduction cascades, which appear to be conserved in all eukaryotes, probably coordinate cytoskeletal reorganization and transcriptional activation via a variety of protein and lipid kinases, and other as-yet-undefined target proteins. The dissection of these cascades has begun with the identification of a growing number of Rho-p21 interacting proteins, though for the most part it remains to be established which of these proteins represents bona fide targets. Here we describe a method that was developed to analyze p21-interacting proteins in total protein extracts after their separation on sodium dodecyl sulfate (SDS)-polyacrylamide gels. In fact, both guanosine 5′ triphosphatase (GTPase)-activating proteins and GTPase-inhibitory proteins can be detected in situ, and the latter class have been successfully identified in expression screens. Novel p21-targets have now been characterized by this method, including the Cdc42-binding tyrosine kinase (ACK) (1); the p21-activated kinase (PAK) (2,3); the p160 RhoA-associated kinase (ROK) (4,5); and the neutrophil Wiscott-Aldrich Syndrome protein (WASP) (6). Deletion analysis of a number of cDNAs encoding these proteins have shown the p2l-binding domains encompass less than 60 amino acid residues (1,2).KeywordsSodium Dodecyl SulfateExpression ScreeningAgarose PlateConserve Amino Acid MotifProtein OverlayThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Bee1, a yeast protein with homology to Wiscott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton.

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of delta bee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that delta bee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain-containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.