Wiskott-Aldrich Syndrome Protein Family Proteins Research Articles

WASP family proteins: Molecular mechanisms and implications in human disease

Proteins of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in regulating actin cytoskeletal dynamics in a wide range of cellular processes. Genetic mutations or misregulation of these proteins are tightly associated with many diseases. The WASP-family proteins act by transmitting various upstream signals to their conserved WH2-Central-Acidic (WCA) peptide sequence at the C-terminus, which in turn binds to the Arp2/3 complex to stimulate the formation of branched actin networks at membranes. Despite this common feature, the regulatory mechanisms and cellular functions of distinct WASP-family proteins are very different. Here, we summarize and clarify our current understanding of WASP-family proteins and how disruption of their functions is related to human disease.

The Role of WAVE2 Signaling in Cancer.

The Wiskott–Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE)—WAVE1, WAVE2 and WAVE3 regulate rapid reorganization of cortical actin filaments and have been shown to form a key link between small GTPases and the actin cytoskeleton. Upon receiving upstream signals from Rho-family GTPases, the WASP and WAVE family proteins play a significant role in polymerization of actin cytoskeleton through activation of actin-related protein 2/3 complex (Arp2/3). The Arp2/3 complex, once activated, forms actin-based membrane protrusions essential for cell migration and cancer cell invasion. Thus, by activation of Arp2/3 complex, the WAVE and WASP family proteins, as part of the WAVE regulatory complex (WRC), have been shown to play a critical role in cancer cell invasion and metastasis, drawing significant research interest over recent years. Several studies have highlighted the potential for targeting the genes encoding either part of or a complete protein from the WASP/WAVE family as therapeutic strategies for preventing the invasion and metastasis of cancer cells. WAVE2 is well documented to be associated with the pathogenesis of several human cancers, including lung, liver, pancreatic, prostate, colorectal and breast cancer, as well as other hematologic malignancies. This review focuses mainly on the role of WAVE2 in the development, invasion and metastasis of different types of cancer. This review also summarizes the molecular mechanisms that regulate the activity of WAVE2, as well as those oncogenic pathways that are regulated by WAVE2 to promote the cancer phenotype. Finally, we discuss potential therapeutic strategies that target WAVE2 or the WAVE regulatory complex, aimed at preventing or inhibiting cancer invasion and metastasis.

Mechanism of WASP and WAVE family proteins in the progression of prostate cancer.

Prostate cancer (PCa) is the second most commonly diagnosed and third lethal cause of death from cancer in men worldwide. Despite the availability of vast treatment procedures, still the high occurrence of invasion and metastasis of PCa are reported in cancer patients. The WASP (Wiskott-Aldrich syndrome protein) and WAVE (WASP family verprolin homologous protein) family of proteins are actin cytoskeleton regulatory proteins, reported to enhance cancer cell invasion and migration in prostate cancer. Hence, this review sheds light on the studies that explored the potential role of WASP and WAVE family of proteins in invasion and metastasis of prostate cancer. The research articles explored for the completion of this review were mostly from PubMed and Google Scholar by using the appropriate keywords for indexing. The conserved function of WASP and WAVE protein family is to receive the upstream signals from the Rho GTPase family and transmit them to activate the Arp2/3 complex that leads to rapid actin polymerization at leading edge of cells, which is crucial for PCa metastasis. Therefore, targeting these proteins could reflect a very interesting therapeutic opportunity to combat prostate cancer.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

Identification of Wiskott-Aldrich syndrome protein (WASP) binding sites on the branched actin filament nucleator Arp2/3 complex

Arp2/3 complex nucleates branched actin filaments important for cellular motility and endocytosis. WASP family proteins are Arp2/3 complex activators that play multiple roles in branching nucleation, but little is known about the structural bases of these WASP functions, owing to an incomplete understanding of how WASP binds Arp2/3 complex. Recent data show WASP binds two sites, and biochemical and structural studies led to models in which the WASP C segment engages the barbed ends of the Arp3 and Arp2 subunits while the WASP A segment binds the back side of the complex on Arp3. However, electron microscopy reconstructions showed density for WASP inconsistent with these models on the opposite (front) side of Arp2/3 complex. Here we use chemical cross-linking and mass spectrometry (XL-MS) along with computational docking and structure-based mutational analysis to map the two WASP binding sites on the complex. Our data corroborate the barbed end and back side binding models and show one WASP binding site on Arp3, on the back side of the complex, and a second site on the bottom of the complex, spanning Arp2 and ARPC1. The XL-MS-identified cross-links rule out the front side binding model and show that the A segment of WASP binds along the bottom side of the ARPC1 subunit, instead of at the Arp2/ARPC1 interface, as suggested by FRET experiments. The identified binding sites support the Arp3 tail release model to explain WASP-mediated activating conformational changes in Arp2/3 complex and provide insight into the roles of WASP in branching nucleation.

Cellular functions of WASP family proteins at a glance.

Proteins of the Wiskott-Aldrich syndrome protein (WASP) family function as nucleation-promoting factors for the ubiquitously expressed Arp2/3 complex, which drives the generation of branched actin filaments. Arp2/3-generated actin regulates diverse cellular processes, including the formation of lamellipodia and filopodia, endocytosis and/or phagocytosis at the plasma membrane, and the generation of cargo-laden vesicles from organelles including the Golgi, endoplasmic reticulum (ER) and the endo-lysosomal network. Recent studies have also identified roles for WASP family members in promoting actin dynamics at the centrosome, influencing nuclear shape and membrane remodeling events leading to the generation of autophagosomes. Interestingly, several WASP family members have also been observed in the nucleus where they directly influence gene expression by serving as molecular platforms for the assembly of epigenetic and transcriptional machinery. In this Cell Science at a Glance article and accompanying poster, we provide an update on the subcellular roles of WHAMM, JMY and WASH (also known as WASHC1), as well as their mechanisms of regulation and emerging functions within the cell.

WASP family proteins, more than Arp2/3 activators.

Wiskott–Aldrich syndrome protein (WASP) family proteins have been extensively characterized as factors that promote the nucleation of actin through the activation of the protein complex Arp2/3. While yeast mostly have a single member of the family, mammalian cells have at least six different members, often with multiple isoforms. Members of the family are characterized by a common structure. Their N-termini are varied and are considered to confer spatial and temporal regulation of Arp2/3-activating activity, whereas their C-terminal half contains a polyproline-rich region, one or more WASP homology-2 (WH2) actin-binding domains and motifs that bind directly to Arp2/3. Recent studies, however, indicate that the yeast WASP homologue Las17 is able to nucleate actin independently of Arp2/3 through the function of novel G-actin-binding activities in its polyproline region. This allows Las17 to generate the mother filaments that are needed for subsequent Arp2/3 recruitment and activation during the actin polymerization that drives endocytic invagination in yeast. In this review, we consider how motifs within the polyproline region of Las17 support nucleation of actin filaments, and whether similar mechanisms might exist among other family members.

WASH has a critical role in NK cell cytotoxicity through Lck-mediated phosphorylation.

Natural killer (NK) cells are important effector cells of the innate immune system to kill certain virus-infected and transformed cells. Wiskott–Aldrich Syndrome protein (WASP) and SCAR homolog (WASH) has been identified as a member of WASP family proteins implicated in regulating the cytoskeletal reorganization, yet little is known about its function in lymphocytes. Here we demonstrate that WASH is crucial for NK cell cytotoxicity. WASH was found to colocalize with lytic granules upon NK cell activation. Knockdown of WASH expression substantially inhibited polarization and release of lytic granules to the immune synapse, resulting in the impairment of NK cell cytotoxicity. More importantly, our data also define a previously unappreciated mechanism for WASH function, in which Src family kinase Lck can interact with WASH and induce WASH phosphorylation. Mutation of tyrosine residue Y141, identified here as the major site of WASH phosphorylation, partially blocked WASH tyrosine phosphorylation and NK cell cytotoxicity. Taken together, these observations suggest that WASH has a pivotal role for regulation of NK cell cytotoxicity through Lck-mediated Y141 tyrosine phosphorylation.

Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3's barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex.

Role of the WASP and WAVE family proteins in breast cancer invasion and metastasis.

The Wiskott–Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) family are a group of molecules that form a key link between GTPases and the actin cytoskeleton. The role of WASP/WAVE family proteins in the control of actin polymerization through activation of the actin-related protein 2/3 complex is critical in the formation of the actin-based membrane protrusions seen in cell migration and invasion. For this reason, the activity of the WASP/WAVE family in cancer cell invasion and migration has been of great interest in recent years. Many reports have highlighted the potential of targeting the WASP/WAVE family as a therapy for the prevention of cancer progression, in particular breast cancer. This review focuses on the role of the WASP/WAVE family in breast cancer cell invasion and migration and how this relates to the molecular mechanisms of WASP/WAVE activity, their exact contributions to the stages of cancer progression, and how this can lead to the development of anticancer drugs that target the WASP/WAVE family and related pathways.

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

Multiple repeat elements within the FAM21 tail link the WASH actin regulatory complex to the retromer

Wiskott-Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E](3-10)-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

WAVE2 Forms a Complex with PKA and Is Involved in PKA Enhancement of Membrane Protrusions

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation.

WASP and WAVE family proteins: Friends or foes in cancer invasion?

Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) family proteins activate cells' major actin nucleating machinery, the actin-related protein 2/3 (Arp2/3) complex, leading to the formation and remodeling of cortical actin filament networks. Cortical actin regulation is critical in many aspects of cell physiology including cell-cell adhesion and cell motility, whose dysregulation is directly associated with cancer invasion and metastasis. In line with this association, the WASP and WAVE family proteins have been reported to be involved in cancer malignancies. What is puzzling, however, is that they can act as either enhancers or suppressors of cancer malignancies depending on the type of cancer and its pathological stage. We are still far from understanding the roles of the WASP and WAVE family proteins in cancer progression. Here, we summarize the recent advances of studies of the WASP and WAVE family proteins with respect to cancer invasion and we offer a model that can account for the diverse outcomes originating from dysregulated WASP and WAVE family proteins in cancer development.

Physical Mechanisms of Signal Integration by WASP Family Proteins

The proteins of the Wiskott-Aldrich syndrome protein (WASP) family are activators of the ubiquitous actin nucleation factor, the Arp2/3 complex. WASP family proteins contain a C-terminal VCA domain that binds and activates the Arp2/3 complex in response to numerous inputs, including Rho family GTPases, phosphoinositide lipids, SH3 domain-containing proteins, kinases, and phosphatases. In the archetypal members of the family, WASP and N-WASP, these signals are integrated through two levels of regulation, an allosteric autoinhibitory interaction, in which the VCA is sequestered from the Arp2/3 complex, and dimerization/oligomerization, in which multi-VCA complexes are better activators of the Arp2/3 complex than monomers. Here, we review the structural, biochemical, and biophysical details of these mechanisms and illustrate how they work together to control WASP activity in response to multiple inputs. These regulatory principles, derived from studies of WASP and N-WASP, are likely to apply broadly across the family.

Contributions of Wiskott–Aldrich syndrome family cytoskeletal regulatory adapters to immune regulation

Cytoskeletal structure and dynamic rearrangement are integrally involved in coupling external stimuli to the orchestrated network of molecular interactions and cellular responses required for T-cell effector function. Members of the Wiskott-Aldrich syndrome protein (WASp) family are now widely recognized as cytoskeletal scaffolding adapters that coordinate the transmission of stimulatory signals to downstream induction of actin remodeling and cytoskeletal-dependent T-cell responses. In this review, we discuss the structural and functional properties of the WASp family members, with an emphasis on the roles of these proteins in the molecular pathways underpinning T-cell activation. The contributions of WASp family proteins and the cytoskeletal reorganization they evoke to expression of specific T-cell effector functions and the implications of such activity to normal immune responses and to the immunologic deficits manifested by Wiskott-Aldrich syndrome patients are also described.

The WASP and WAVE family proteins

A review of the cytoskeleton-organizing WASP and WAVE family proteins.

The WASP–WAVE protein network: connecting the membrane to the cytoskeleton

Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins are scaffolds that link upstream signals to the activation of the ARP2/3 complex, leading to a burst of actin polymerization. ARP2/3-complex-mediated actin polymerization is crucial for the reorganization of the actin cytoskeleton at the cell cortex for processes such as cell movement, vesicular trafficking and pathogen infection. Large families of membrane-binding proteins were recently found to interact with WASP and WAVE family proteins, therefore providing a new layer of membrane-dependent regulation of actin polymerization.

WASP Family Proteins Act between Cytoskeleton and Cellular Signaling Pathways

This review considers the proteins of the WASP (Wiskott-Aldrich syndrome protein) family and their role in the regulation of actin-based motility. It contains detailed classification of the WASP family proteins and data on their subcellular localization. Impairments of expression of the WASP family proteins cause certain cell pathologies. The review also deals with domain organization of these proteins and proteins interacting with various domains of the WASP proteins. Special attention is given to analysis of the role of the WASP family proteins in initiating directed actin assembly in the leading edge of the migrating cell and on the surface of some bacteria. Putative pathways of regulation of WASP proteins by various protein ligands and their links with cell signaling systems are considered.