WASP-interacting Protein Research Articles

A triple-pose complex between an extended WIP motif and a C-terminal SH3 domain modulates cortactin activity.

The central domain of WASp-interacting protein (WIP) interacts with the cortactin SH3 domain through a previously undefined binding motif. This interaction affects extracellular matrix (ECM) degradation and the invasive phenotype of cells. Here, using NMR-based methods, we identify the major WIP epitope modulating this binding event as residues 168-183, an unexpectedly long segment uncharacteristic of SH3 peptidic ligands. A scanning mutagenesis analysis showed that peptide binding 'hotspots' are distributed throughout the binding sequence. To uncover the structural basis of WIP-cortactin recognition we utilized edited-filtered NOESY experiments to determine the structure of the intermediate-affinity SH3/peptide complex. Analysis of the NOESY pattern suggests that the peptide sequence dictates three interchanging binding modes, two oppositely oriented canonical poses involving N-terminal interactions, corresponding to class I and class II complexes, and a non-canonical pseudo-class II pose involving C-terminal interactions. The latter pose highlights the importance of the hydrophobic surface adjacent to the canonical binding grooves and accounts for the extended binding motif. Design of mutant peptides with increased affinity based on this multi-conformational complex demonstrates how these structural insights may impact design of improved inhibitors of the WIP-cortactin interaction with potential therapeutic applications.

Multifaceted role of the actin-binding protein WIP: Promotor and inhibitor of tumor progression and dissemination.

Cancer cells depend on actin cytoskeleton reorganization to achieve hallmark malignant functions including abnormal activation, proliferation, migration and invasiveness. (Neural)-Wiskott-Aldrich Syndrome protein ((N-)WASP) binds actin and forms a complex with the WASP-interacting protein (WIP), which plays a critical role in regulating the actin cytoskeleton, through (N)-WASP-dependent and independent functions. Mutations in the WIP gene (WIPF1) lead to severe early onset immunodeficiency in humans and severe autoimmunity and shortened lifespan in mice. This review covers the available evidence about the physiological role of WIP in different tissues and its contribution to human disease, focusing on cancer. In solid tumors overexpression of WIP has mostly been associated with tumor initiation, progression and dissemination through matrix degradation by invadopodia, while a suppressive function has been shown for WIP in certain hematological cancers. Interestingly, a minority of studies suggest a protective role for WIP in specific tumor contexts. These data support the need for further research to fully understand the mechanisms underlying WIP's diverse functions in health and disease and raise important questions for future work.

WIPF1 promotes gastric cancer progression by regulating PI3K/Akt signaling in a myocardin-dependent manner

Wiskott-Aldrich syndrome protein-interacting protein family member 1 (WIPF1) is associated with malignant tumor progression. However, molecular links between WIPF1 and gastric cancer (GC) remain elusive. The expression of WIPF1 was detected in GC tissues and cells. WIPF1 was overexpressed in GC tissues and cells and high expression of WIPF1 was an independent risk factor for a poor prognosis in patients with GC. Further experiments indicated that WIPF1 promoted the proliferation, invasion, and migration of GC cells invivo and invitro. WIPF1-regulated genes were closely related to cell proliferation and migration in GC, and silencing WIPF1 significantly repressed PI3K/AKT signaling pathway activation. WIPF1 was activated by myocardin (MYOCD) translation. Rescue experiments confirmed that MYOCD promotes the proliferation, invasion, and migration of GC cells in a WIPF1-dependent manner and activates the PI3K/AKT signaling pathway. MYOCD may transactivate WIPF1 and facilitate GC cell growth and metastasis by activating the PI3K/AKT signaling pathway.

Indoxyl sulfate- and P-cresol-induced monocyte adhesion and migration is mediated by integrin-linked kinase-dependent podosome formation

Cardiovascular disease is an important cause of death in patients with chronic kidney disease (CKD). Protein-bound uremic toxins, such as p-cresyl and indoxyl sulfate (IS), are poorly removed during hemodialysis, leading to vascular endothelial dysfunction and leukocyte extravasation. These processes can be related to dynamic adhesion structures called podosomes. Several studies have indicated the role of integrin-linked kinase (ILK) in the accumulation of integrin-associated proteins in podosomes. Here, we investigated the involvement of ILK and podosome formation in the adhesion and extravasation of monocytes under p-cresol (pc) and IS exposure. Incubation of THP-1 human monocyte cells with these toxins upregulated ILK kinase activity. Together, both toxins increased cell adhesion, podosome formation, extracellular matrix degradation, and migration of THP-1 cells, whereas ILK depletion with specific small interfering RNAs suppressed these processes. Interestingly, F-actin colocalized with cortactin in podosome cores, while ILK was colocalized in podosome rings under toxin stimulation. Podosome Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) and AKT protein depletion demonstrated that monocyte adhesion depends on podosome formation and that the ILK/AKT signaling pathway is involved in these processes. Ex vivo experiments showed that both toxins induced adhesion and podosome formation in leukocytes from wild-type mice, whereas these effects were not observed in leukocytes of conditional ILK-knockdown animals. In summary, under pc and IS stimulation, monocytes increase podosome formation and transmigratory capacity through an ILK/AKT signaling pathway-dependent mechanism, which could lead to vascular injury. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

Structure and fuzziness in the WIP-cortactin regulatory complex

WASp Interacting Protein (WIP) is a cellular multi-tasker that regulates actin cytoskeleton and remodeling, and an important player in a range of protein-protein interactions impacting health and disease. One of its central binding partners is cortactin, a key nucleation-promoting factor responsible for actin polymerization and a biomarker for invasive cancers. While it has been suggested that the WIP-cortactin interaction is mediated by the proline-rich region of WIP, this hypothesis was not supported by structural evidence.

WIP, YAP/TAZ and Actin Connections Orchestrate Development and Transformation in the Central Nervous System

YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are transcription co-regulators that make up the terminal components of the Hippo signaling pathway, which plays a role in organ size control and derived tissue homeostasis through regulation of the proliferation, differentiation and apoptosis of a wide variety of differentiated and stem cells. Hippo/YAP signaling contributes to normal development of the nervous system, as it participates in self-renewal of neural stem cells, proliferation of neural progenitor cells and differentiation, activation and myelination of glial cells. Not surprisingly, alterations in this pathway underlie the development of severe neurological diseases. In glioblastomas, YAP and TAZ levels directly correlate with the amount of the actin-binding molecule WIP (WASP interacting protein), which regulates stemness and invasiveness. In neurons, WIP modulates cytoskeleton dynamics through actin polymerization/depolymerization and acts as a negative regulator of neuritogenesis, dendrite branching and dendritic spine formation. Our working hypothesis is that WIP regulates the YAP/TAZ pools using a Hippo-independent pathway. Thus, in this review we will present some of the data that links WIP, YAP and TAZ, with a focus on their function in cells from the central and peripheral nervous systems. It is hoped that a better understanding of the mechanisms involved in brain and nervous development and the pathologies that arise due to their alteration will reveal novel therapeutic targets for neurologic diseases.

WIP Modulates Oxidative Stress through NRF2/KEAP1 in Glioblastoma Cells.

Due to their high metabolic rate, tumor cells produce exacerbated levels of reactive oxygen species that need to be under control. Wiskott–Aldrich syndrome protein (WASP)-interacting protein (WIP) is a scaffold protein with multiple yet poorly understood functions that participates in tumor progression and promotes cancer cell survival. However, its participation in the control of oxidative stress has not been addressed yet. We show that WIP depletion increases the levels of reactive oxygen species and reduces the levels of transcription factor NRF2, the master regulator of redox homeostasis. We found that WIP stabilizes NRF2 by restraining the activity of its main NRF2 repressor, the E3 ligase adapter KEAP1, because the overexpression of a NRF2ΔETGE mutant that is resistant to targeted proteasome degradation by KEAP1 or the knock-down of KEAP1 maintains NRF2 levels in the absence of WIP. Mechanistically, we show that the increased KEAP1 activity in WIP-depleted cells is not due to the protection of KEAP1 from autophagic degradation, but is dependent on the organization of the Actin cytoskeleton, probably through binding between KEAP1 and F-Actin. Our study provides a new role of WIP in maintaining the oxidant tolerance of cancer cells that may have therapeutic implications.

The Disordered Cellular Multi-Tasker WIP and Its Protein-Protein Interactions: A Structural View.

WASp-interacting protein (WIP), a regulator of actin cytoskeleton assembly and remodeling, is a cellular multi-tasker and a key member of a network of protein–protein interactions, with significant impact on health and disease. Here, we attempt to complement the well-established understanding of WIP function from cell biology studies, summarized in several reviews, with a structural description of WIP interactions, highlighting works that present a molecular view of WIP’s protein–protein interactions. This provides a deeper understanding of the mechanisms by which WIP mediates its biological functions. The fully disordered WIP also serves as an intriguing example of how intrinsically disordered proteins (IDPs) exert their function. WIP consists of consecutive small functional domains and motifs that interact with a host of cellular partners, with a striking preponderance of proline-rich motif capable of interactions with several well-recognized binding partners; indeed, over 30% of the WIP primary structure are proline residues. We focus on the binding motifs and binding interfaces of three important WIP segments, the actin-binding N-terminal domain, the central domain that binds SH3 domains of various interaction partners, and the WASp-binding C-terminal domain. Beyond the obvious importance of a more fundamental understanding of the biology of this central cellular player, this approach carries an immediate and highly beneficial effect on drug-design efforts targeting WIP and its binding partners. These factors make the value of such structural studies, challenging as they are, readily apparent.

Loss of Wasl improves pancreatic cancer outcome.

Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about its function in pancreatic ductal adenocarcinoma (PDAC). In this study, we performed in silico analysis of WASL expression in PDAC patients and found a correlation between low WASL expression and prolonged survival. To clarify the role of Wasl in pancreatic carcinogenesis, we used 2 oncogenic Kras-based PDAC mouse models with pancreas-specific Wasl deletion. In line with human data, both mouse models had an increased survival benefit due to either impaired tumor development in the presence of the tumor suppressor Trp53 or the delayed tumor progression and senescent phenotype upon genetic ablation of Trp53. Mechanistically, loss of Wasl resulted in cell-autonomous senescence through displacement of the N-WASP binding partners WASP-interacting protein (WIP) and p120ctn; vesicular accumulation of GSK3β, as well as YAP1 and phosphorylated β-catenin, which are components of the destruction complex; and upregulation of Cdkn1a(p21), a master regulator of senescence. Our findings, thus, indicate that Wasl functions in an oncogenic manner in PDAC by promoting the deregulation of the p120-catenin/β-catenin/p21 pathway. Therefore, strategies to reduce N-WASP activity might improve the survival outcomes of PDAC patients.

WASP and Mst1 coregulate B-cell development and B-cell receptor signaling

AbstractMst1 is a serine/threonine kinase involved in cell survival, proliferation, apoptosis, and tumorigenesis. In mice, Mst1 regulates actin dynamics required for T-cell adhesion and migration, which correlate with thymic egress and entry into lymphatic tissue. The role of Mst1 in B cells and how it may control actin-dependent processes has not been well characterized. Wiskott-Aldrich syndrome protein (WASP) deficiency only moderately affects development and B-cell receptor (BCR) signaling, suggesting WASP likely associates with other molecules. We investigated whether Mst1 associates with WASP to regulate B-cell development and activation. Experimenting on Mst1/WASP double knockout (DKO) mice, we found a severe defect in the bone marrow B-cell development, and BCR signaling in the DKO mice was severely reduced. Even though WASP or Mst1 could influence the early B-cell activation, we found that the early activation events such as B-cell spreading, BCR clustering, and BCR signaling were much more impaired in the B cells from DKO mice. Furthermore, reciprocal regulation between Mst1 and WASP was observed in WASP and Mst1 KO mice, whereby the localization and function of phosphorylated WASP were affected in Mst1 KO mice. Most importantly, Mst1 inhibits the expression of WASP by decreasing the expression of WASP-interacting protein. Interestingly, we also found that WASP deficiency in patients and mice interferes with phosphorylated Mst1 localization and therefore function in B cells. Overall, our study provides a partner for WASP to regulate B-cell development and BCR signaling, as well as the reciprocal regulating molecular mechanism of one another.

Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish.

Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/β-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/β-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/β-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.

Wiskott-Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma.

In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-β. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.

The Lack of WIP Binding to Actin Results in Impaired B Cell Migration and Altered Humoral Immune Responses

SummaryWiskott-Aldrich syndrome protein (WASp) is a main cytoskeletal regulator in B cells. WASp-interacting protein (WIP) binds to and stabilizes WASp but also interacts with actin. Using mice with a mutated actin binding domain of WIP (WIPΔABD), we here investigated the role of WIP binding to actin during B cell activation. We found an altered differentiation of WIPΔABD B cells and diminished antibody affinity maturation after immunization. Mechanistically, WIPΔABD B cells showed impaired B cell receptor (BCR)-induced PI3K signaling and actin reorganization, likely caused by diminished CD81 expression and altered CD19 dynamics on the B cell surface. WIPΔABD B cells displayed reduced in vivo motility, concomitantly with impaired chemotaxis and defective F-actin polarization, HS1 phosphorylation, and polarization of HS1 to F-actin-rich structures after CXCL12 stimulation in vitro. We thus concluded that WIP binding to actin, independent of its binding to WASp, is critical for actin cytoskeleton plasticity in B cells.

WIP/ITSN1 complex is involved in cellular vesicle trafficking and formation of filopodia-like protrusions

WIP (WASP interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) regulates actin polymerization that is crucial for invadopodia and filopodia formation. Recently, we reported the WIP interaction with ITSN1 which is highly implicated in endo-/exocytosis, apoptosis, mitogenic signaling and cytoskeleton rearrangements. Here we demonstrate that the WIP/ITSN1 complex is involved in the transferrin receptor recycling and partially co-localizes with a marker of the fast recycling endosomes, RAB4. Moreover, ITSN1 recruits WIP to RAB4-positive vesicles upon overexpression. Our data indicate that WIP enhances the interaction of N-WASP with ITSN1 and promotes ITSN1/β-actin association. Moreover, the WIP/ITSN1-L complex facilitates formation of filopodia-like protrusions in MCF-7 cells. Thus, WIP/ITSN1 complex is involved in the cellular vesicle trafficking and actin-dependent membrane processes.

WIP-YAP/TAZ as A New Pro-Oncogenic Pathway in Glioma.

Wild-type p53 (wtp53) is described as a tumour suppressor gene, and mutations in p53 occur in many human cancers. Indeed, in high-grade malignant glioma, numerous molecular genetics studies have established central roles of RTK-PI3K-PTEN and ARF-MDM2-p53 INK4a-RB pathways in promoting oncogenic capacity. Deregulation of these signalling pathways, among others, drives changes in the glial/stem cell state and environment that permit autonomous growth. The initially transformed cell may undergo subsequent modifications, acquiring a more complete tumour-initiating phenotype responsible for disease advancement to stages that are more aggressive. We recently established that the oncogenic activity of mutant p53 (mtp53) is driven by the actin cytoskeleton-associated protein WIP (WASP-interacting protein), correlated with tumour growth, and more importantly that both proteins are responsible for the tumour-initiating cell phenotype. We reported that WIP knockdown in mtp53-expressing glioblastoma greatly reduced proliferation and growth capacity of cancer stem cell (CSC)-like cells and decreased CSC-like markers, such as hyaluronic acid receptor (CD44), prominin-1 (CD133), yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). We thus propose a new CSC signalling pathway downstream of mtp53 in which Akt regulates WIP and controls YAP/TAZ stability. WIP drives a mechanism that stimulates growth signals, promoting YAP/TAZ and β-catenin stability in a Hippo-independent fashion, which allows cells to coordinate processes such as proliferation, stemness and invasiveness, which are key factors in cancer progression. Based on this multistep tumourigenic model, it is tantalizing to propose that WIP inhibitors may be applied as an effective anti-cancer therapy.

From Disordered Polypeptide to Functional Regulator: A Structural View of WASp-Interacting Protein and its Complex with WASp in Human T-Cells

From Disordered Polypeptide to Functional Regulator: A Structural View of WASp-Interacting Protein and its Complex with WASp in Human T-Cells

Tuning of in vivo cognate B-T cell interactions by Intersectin 2 is required for effective anti-viral B cell immunity.

Wiskott-Aldrich syndrome (WAS) is an immune pathology associated with mutations in WAS protein (WASp) or in WASp interacting protein (WIP). Together with the small GTPase Cdc42 and other effectors, these proteins participate in the remodelling of the actin network downstream of BCR engagement. Here we show that mice lacking the adaptor protein ITSN2, a G-nucleotide exchange factor (GEF) for Cdc42 that also interacts with WASp and WIP, exhibited increased mortality during primary infection, incomplete protection after Flu vaccination, reduced germinal centre formation and impaired antibody responses to vaccination. These defects were found, at least in part, to be intrinsic to the B cell compartment. In vivo, ITSN2 deficient B cells show a reduction in the expression of SLAM, CD84 or ICOSL that correlates with a diminished ability to form long term conjugates with T cells, to proliferate in vivo, and to differentiate into germinal centre cells. In conclusion, our study not only revealed a key role for ITSN2 as an important regulator of adaptive immune-response during vaccination and viral infection but it is also likely to contribute to a better understanding of human immune pathologies.

Backbone chemical shifts for the complex between WASp interacting protein (WIP) and the EVH1 domain of WASp

Backbone chemical shifts for the complex between WASp interacting protein (WIP) and the EVH1 domain of WASp

New Structural Insights into Formation of the Key Actin Regulating WIP-WASp Complex Determined by NMR and Molecular Imaging.

Wiskott-Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells and responsible for actin-dependent processes, including cellular activation, migration, and invasiveness. The C-terminal domain of WASp-Interacting Protein (WIP) binds to WASp and regulates its activity by shielding it from degradation in a phosphorylation dependent manner as we previously demonstrated. Mutations in the WAS-encoding gene lead to the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Here, we shed a first structural light upon this function of WIP using nuclear magnetic resonance (NMR) and in vivo molecular imaging. Coexpression of fragments WASp(20-158) and WIP(442-492) allowed the purification and structural characterization of a natively folded complex, determined to form a characteristic pleckstrin homology domain with a mixed α/β-fold and central two-winged β-sheet. The WIP-derived peptide, unstructured in its free form, wraps around and interacts with WASp through short structural elements. Förster resonance energy transfer (FRET) and biochemical experiments demonstrated that, of these elements, WIP residues 454-456 are the major contributor to WASp affinity, and the previously overlooked residues 449-451 were found to have the largest effect upon WASp ubiquitylation and, presumably, degradation. Results obtained from this complementary combination of technologies link WIP-WASp affinity to protection from degradation. Our findings about the nature of WIP·WASp complex formation are relevant for ongoing efforts to understand hematopoietic cell behavior, paving the way for new therapeutic approaches to WAS and XLT.

Mutant p53 oncogenic functions in cancer stem cells are regulated by WIP through YAP/TAZ.

Wild-type p53 (wtp53) is described as a tumour suppressor gene; mutations in this gene occur in many human cancers and promote oncogenic capacity. Here, we establish that the oncogenic activity of mutant p53 (mtp53) is driven by the WASP-interacting protein (WIP). WIP knockdown from mtp53-expressing glioblastoma and breast cancer cells (BCC) greatly reduced proliferation and growth capacity of cancer stem cell (CSC)-like cells and decreased CSC-like markers (CD133, CD44 or YAP/TAZ). mtp53 overexpression in human astrocytes enhanced their proliferative capacity in suspension culture and increased expression of CSC markers and WIP. WIP knockdown compromised tumour glioblastoma and BCC growth capacity in vivo. We show that WIP is phosphorylated by AKT2 and is regulated by mtp53/p63 through enhancement of PI3K/AKT2-mediated integrin/receptor recycling pathways. WIP regulates this oncogenic pathway by controlling YAP/TAZ stability. We thus establish a new CSC signalling pathway downstream of mtp53 in which AKT2 regulates WIP and controls YAP/TAZ stability.