Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system, a proline-rich WASP-interacting protein (WIP) has been identified that coimmunoprecipitates with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and includes actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increases polymerized actin content and induces the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function (Ramesh, 1997).
Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signaling cascades to actin polymerization. The amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP, is responsible for N-WASP recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella, WIP is recruited by N-WASP. These observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signaling pathways to recruit the N-WASP-WIP complex. It is proposed that the N-WASP-WIP complex has a pivotal function in integrating signaling cascades that lead to actin polymerization (Moreau, 2000).
Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant [Cdc42(V12)] and by N-WASP. These results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella (Martinez-Quiles, 2001).
A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, the exact nature of its binding site has not been known. The N-WASP WH1 binding motif in WIP has now been identified and characterized in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand. Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif is conserved in WIP, CR16, WICH, and yeast verprolin (Zett, 2002).
The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells. Given that 80% of identified Wiskott-Aldrich Syndrome patients result from mutations in the binding site for WASP-interacting-protein (WIP), the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs) was examined. The results show that WIP is essential both for the formation of actin cores containing WASP and cortactin and for the organization of integrin and integrin-associated proteins in circular arrays, specific characteristics of podosome structure. It was also found that WIP is essential for the maintenance of the high turnover of adhesions and polarity in DCs. WIP exerts these functions by regulating calpain-mediated cleavage of WASP and by facilitating the localization of WASP to sites of actin polymerization at podosomes. Taken together, these results indicate that WIP is critical for the regulation of both the stability and localization of WASP in migrating DCs and suggest that WASP and WIP operate as a functional unit to control DC motility in response to changes in the extracellular environment (Chou, 2006).
Missense mutants that cause the immune disorder Wiskott-Aldrich Syndrome (WAS) map primarily to the Enabled/VASP homology 1 (EVH1) domain of the actin regulatory protein WASP. This domain has been implicated in both peptide and phospholipid binding. N-WASP EVH1 domain does not bind phosphatidyl inositol-(4,5)-bisphosphate, as previously reported, but does specifically bind a 25 residue motif from the WASP Interacting Protein (WIP). The NMR structure of the complex reveals a novel recognition mechanism-the WIP ligand, which is far longer than canonical EVH1 ligands, wraps around the domain, contacting a narrow but extended surface. This recognition mechanism provides a basis for understanding the effects of mutations that cause WAS (Volkman, 2002).
The WASP-interacting protein (WIP) targets WASP/WAVE proteins through a constitutive interaction with an amino-terminal enabled/VASP homology (EVH1) domain. Two distinct N-WASP binding motifs corresponding to WIP residues 451-461 and 461-485, and the structure of a complex between WIP-(461-485) and the N-WASP EVH1 domain have been determined. When combined, the WIP-(451-485) sequence wraps further around the EVH1 domain, extending the interface observed previously. Specific contacts with three WIP epitopes correspond to regions of high sequence conservation in the verprolin family. A central polyproline motif occupies the canonical binding site but in a reversed orientation relative to other EVH1 complexes. This interaction is augmented in the amino- and carboxyl-terminal directions by additional hydrophobic contacts involving WIP residues 454-459 and 475-478, respectively. Disruption of any of the three WIP epitopes reduces N-WASP binding in cells, demonstrating a functional requirement for the entire binding domain, which is significantly longer than the polyproline motifs recognized by other EVH1 domains (Peterson, 2007).
Modulation of actin cytoskeleton assembly is an integral step in many cellular events. A key regulator of actin polymerization is Arp2/3 complex. Cortactin (see Drosophila Cortactin), an F-actin binding protein that localizes to membrane ruffles, is an activator of Arp2/3 complex. A yeast two-hybrid screen revealed the interaction of the cortactin Src homology 3 (SH3) domain with a peptide fragment derived from a cDNA encoding a region of WASp-Interacting Protein (WIP). GST-cortactin interacts with WIP in an SH3-dependent manner. The subcellular localization of cortactin and WIP coincides at the cell periphery. WIP increases the efficiency of cortactin-mediated Arp2/3 complex activation of actin polymerization in a concentration-dependent manner. Lastly, coexpression of cortactin and WIP stimulated membrane protrusions. It is concluded that WIP, a protein involved in filopodia formation, binds to both actin monomers and cortactin. Thus, recruitment of actin monomers to a cortactin-activated Arp2/3 complex likely leads to the observed increase in cortactin activation of Arp2/3 complex by WIP. These data suggest that a cortactin-WIP complex functions in regulating actin-based structures at the cell periphery (Kinley, 2003).
An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway is described. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome (Ho, 2004).
Sequence analysis reveals that Toca-1 is structurally related to proteins of the PCH (pombe Cdc15 h) family; these proteins have been implicated recently in a wide variety of actin-dependent processes, including cytokinesis, membrane trafficking, and cellular morphogenesis. This protein family is conserved throughout eukaryotic evolution and includes human formin binding protein 17 (FBP17), human Cdc42-interacting protein 4 (CIP4), human syndapins (see Drosophila Syndapin), D. melanogaster Cip4, C. elegans CE27939, S. cerevisiae Bzz1p, and S. pombe Cdc15. Members of this protein family are defined by a common domain structure that includes a FER/CIP4 homology (FCH) domain at the N terminus and one or two Src homology 3 (SH3) domains at the C terminus. The FCH domain is found in a large number of proteins involved in signal transduction, but its function is largely unknown. In addition, many PCH proteins are also predicted to contain coiled-coil domains. In the case of Toca-1, FBP17, CIP4, and D. melanogaster Cip4, one of these coiled-coil regions has homology to a domain called HR1 (protein kinase C-related kinase homology region 1), which was originally identified as a Rho-interactive module in several RhoA binding proteins. The functional conservation of Toca-1 across species is highlighted by the finding that Toca-1 homologs from X. tropicalis and D. melanogaster can complement the MCAP2B activity in an in vitro assay system (Ho, 2004).
The following view of the Cdc42 signaling pathway is proposed. Formation of PIP2 on membranes (such as a vesicle surface) leads to the recruitment and activation of Cdc42. Prenylated Cdc42 inserts into the membrane and forms high avidity sites that recruit Toca-1 and the N-WASP-WIP complex. Activation of N-WASP then could proceed through one of two paths: both Cdc42 and Toca-1 could cooperate to activate the N-WASP-WIP complex, or Toca-1 could function indirectly by relieving the inhibition of N-WASP by WIP. Toca-1 is ideally positioned to be an important regulatory node for the Cdc42 pathway. The function of Toca-1 suggests a specific mechanism by which PCH family proteins can influence actin nucleation in a wide variety of cellular processes such as vesicle motility and cytokinesis. Important future questions include the precise biochemical mechanism by which the N-WASP-WIP complex is activated by Toca-1 and Cdc42, as well as investigation into the regulation of Toca-1 itself by other signals (Ho, 2004).
Nck is a ubiquitous adaptor molecule composed of three Src homology 3 (SH3) domains followed by a single SH2 domain. Nck links, via its SH2 domain, tyrosine-phosphorylated receptors to effector proteins that contain SH3-binding proline-rich sequences. Recombinant Nck precipitates endogenous WIP, a novel proline-rich protein that interacts with the Wiskott-Aldrich syndrome protein (WASP), from BJAB cell lysates. Nck binds through its second SH3 domain to WIP, and Nck binds to WIP at a site (amino acids 321-415) that differs from the WASP-binding site (amino acids 416-488). WIP has been shown to associate with the actin polymerization regulatory protein profilin and to induce actin polymerization and cytoskeletal reorganization in lymphoid cells. The presence of profilin in Nck precipitates has been demonstrated suggesting that Nck may couple extracellular signals to the cytoskeleton via its interaction with WIP and profilin (Anton, 1998).
The importance of the SH3 domain of Src family kinase Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, mass spectrometry was used to identify approximately 25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). Focus was also placed on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, it was confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. Hck and ELMO1 interact in intact cells and ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. These results suggest that these proteins may be novel activators/effectors of Hck (Scott, 2002).
The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation (Myers, 2006).
WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologs, the function of WIP was analyzed in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome (Vadula, 1999).
Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. Yeast WASP initiates actin assembly at endocytic sites and this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization (Sun, 2006).
Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, WASP-interacting protein homologue (WIP)-1 was identified in C. elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and wsp-1(RNAi) treatment decreased levels of WIP-1 protein. However, wsp-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in wsp-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for wsp-1. Immunostaining showed that WIP-1 is expressed by migrating hypodermal cells, as is WSP-1. This expression during ventral enclosure is reduced in wip-1(RNAi)-treated embryos and wsp-1(RNAi)-treated embryos. These results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1 (Sawa, 2006).
F-actin polymerization following engagement of the T cell receptor (TCR) is dependent on WASP and is critical for T cell activation. The link between TCR and WASP is not fully understood. In resting cells, WASP exists in a complex with WIP, which inhibits its activation by Cdc42. Adaptor protein CrkL binds directly to WIP. Further, TCR ligation results in the formation of a ZAP-70-CrkL-WIP-WASP complex, which is recruited to lipid rafts and the immunological synapse. TCR engagement also causes PKCtheta-dependent phosphorylation of WIP, causing the disengagement of WASP from the WIP-WASP complex, thereby releasing it from WIP inhibition. These results suggest that the ZAP-70-CrkL-WIP pathway and PKCtheta link TCR to WASP activation (Sasahara, 2003).
Wiskott-Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, are severely diminished in T cells from WIP(-/-) mice and are increased by introduction of WIP in these cells. The WASP binding domain of WIP protects WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increases WASP levels in T cells from WIP(-/-) mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP(-/-) mice. Despite its ability to increase WASP levels proteasome inhibition ddoes not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function (de la Fuente, 2007).
The Wiskott-Aldrich syndrome protein (WASP) is a key molecule for transduction of extracellular signals that induce a variety of critical biological events involving actin cytoskeleton rearrangement. Among the cellular partners of WASP, the Wiskott-Aldrich syndrome protein-interacting protein (WIP) has been speculated to play a critical role in the pathophysiology of Wiskott-Aldrich syndrome since WASP mutation hot spots map to the WIP-binding region. The notion that WIP promotes WASP function, however, conflicts with evidence that WIP inhibits WASP-mediated actin polymerization and IL-2 production and suggests a complex regulation of WASP function by WIP. This study shows that WASP gene transfer results in high WASP expression only when WIP is concomitantly expressed in K562 cells. Furthermore, WIP-knockdown experiments demonstrated that T cells with reduced WIP expression show a concordant reduction of WASP levels. Mapping studies using WIP mutants showed that the minimal WIP region able to rescue WASP expression in WIP-knockdown cells was the WASP-binding domain. However, expression of such a minimal domain of WIP failed to rescue WASP-dependent, nuclear factor of activated T-cells-mediated IL-2 transcriptional activity. These results demonstrate that expression of WIP is necessary for functional WASP expression in human cells and provide a new paradigm for understanding the function of these two molecules (Konno, 2007).
The tumor natural killer (NK) cell line YTS was used to examine the cytoskeletal rearrangements required for cytolysis. A multiprotein complex weighing approximately 1.3 mD and consisting of WASp-interacting protein (WIP), Wiskott-Aldrich syndrome protein (WASp), actin, and myosin IIA that formed during NK cell activation was identified. After induction of an inhibitory signal, the recruitment of actin and myosin IIA to a constitutive WIP-WASp complex was greatly decreased. Both actin and myosin IIA were recruited to WIP in the absence of WASp. This recruitment correlated with increased WIP phosphorylation, which was mediated by PKCtheta. Furthermore, the disruption of WIP expression by WIP RNA interference prevented the formation of this protein complex and led to almost complete inhibition of cytotoxic activity. Thus, the multiprotein complex is important for NK cell function, killer cell immunoglobulin-like receptor inhibitory signaling affects proteins involved in cytoskeletal rearrangements, and WIP plays a central role in the formation of the complex and in the regulation of NK cell activity (Krzewski, 2006).
Chemotactic migration of macrophages is critical for the recruitment of leukocytes to inflamed tissues. Macrophages use a specialized adhesive structure called a podosome to migrate. Podosome formation requires the Wiskott-Aldrich syndrome protein (WASP), which is a product of the gene defective in an X-linked inherited immunodeficiency disorder, the Wiskott-Aldrich syndrome. Macrophages from WASP-deficient Wiskott-Aldrich syndrome patients lack podosomes, resulting in defective chemotactic migration. However, the molecular basis for podosome formation is not fully understood. The WASP interacting protein (WIP), a binding partner of WASP, plays an important role in podosome formation in macrophages. WASP binds WIP to form a complex at podosomes, and the knockdown of WIP impairs podosome formation. When WASP binding to WIP is blocked, podosome formation is also impaired. When WASP expression is reduced by small interfering RNA transfection, the amount of the complex of WASP with WIP decreases, resulting in reduced podosome formation. Podosomes are restored by reconstitution of the WASP-WIP complex in WASP knockdown cells. These results indicate that the WASP-WIP complex is required for podosome formation in macrophages. When podosome formation is reduced by blocking WASP binding to WIP, transendothelial migration of macrophages, the most crucial process in macrophage trafficking, is impaired. These results suggest that a complex of WASP with WIP plays a critical role in podosome formation, thereby mediating efficient transendothelial migration of macrophages (Tsuboi, 2007).
In cancer, the deregulation of growth signaling pathways drives changes in the cell's architecture and its environment that allow autonomous growth of tumors. These cells then acquire a tumor-initiating "stemness" phenotype responsible for disease advancement to more aggressive stages. This study shows that high levels of the actin cytoskeleton-associated protein WIP (WASP-interacting protein; see Drosophila Verprolin) correlates with tumor growth, both of which are linked to the tumor-initiating cell phenotype. WIP controls tumor growth by boosting signals that stabilize the YAP/TAZ complex (see Drosophila Yorkie) via a mechanism mediated by the endocytic/endosomal system. When WIP levels are high, the β-catenin Adenomatous polyposis coli (APC)-axin-GSK3 destruction complex (see Drosophila Apc) is sequestered to the multi-vesicular body compartment, where its capacity to degrade YAP/TAZ is inhibited. YAP/TAZ stability is dependent on Rac (see Drosophila Rac1), p21-activated kinase (PAK) and mammalian diaphanous-related formin (mDia; see Drosophila Diaphanous), and is Hippo independent. This close biochemical relationship indicates an oncogenic role for WIP in the physiology of cancer pathology by increasing YAP/TAZ stability (Gargini, 2016).
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