Tyrosine phosphorylation of WIP releases bound WASP and impairs podosome assembly in macrophages.

Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of podosomes is based on a WASP- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and they co-localise in podosome cores. Here, we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP-WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP-WASP binding, cellular WASP is rapidly degraded, leading to disruption of podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP-WASP complex remains intact and podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Bruton's tyrosine kinase (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor.

WIP modulates dendritic spine actin cytoskeleton by transcriptional control of lipid metabolic enzymes.

We identify Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) as a novel component of neuronal synapses whose absence increases dendritic spine size and filamentous actin levels in an N-WASP/Arp2/3-independent, RhoA/ROCK/profilinIIa-dependent manner. These effects depend on the reduction of membrane sphingomyelin (SM) due to transcriptional upregulation of neutral sphingomyelinase (NSM) through active RhoA; this enhances RhoA binding to the membrane, raft partitioning and activation in steady state but prevents RhoA changes in response to stimulus. Inhibition of NSM or SM addition reverses RhoA, filamentous actin and functional anomalies in synapses lacking WIP. Our findings characterize WIP as a link between membrane lipid composition and actin cytoskeleton at dendritic spines. They also contribute to explain cognitive deficits shared by individuals bearing mutations in the region assigned to the gene encoding for WIP.

Cancer stem cell-like phenotype and survival are coordinately regulated by Akt/FoxO/Bim pathway.

Many solid tumors contain a subpopulation of cells with stem characteristics and these are known as cancer stem cells (CSCs) or tumor-initiating cells (TICs). These cells drive tumor growth and appear to be regulated by molecular pathway different from other cells in the tumor bulk. Here, we set out to determine whether elements of the PI3K-AKT pathway are necessary to maintain the CSC-like phenotype in breast tumor cells and for these cells to survive, bearing in mind that the identification of such elements is likely to be relevant to define future therapeutic targets. Our results demonstrate a close relationship between the maintenance of the CSC-like phenotype and the survival of these TICs. Inhibiting PI3K activity, or eliminating AKT activity, mostly that of the AKT1 isoform, produces a clear drop in TICs survival, and a reduction in the generation and growth of CD44(High) /CD24(Low) mammospheres. Surprisingly, the apoptosis of these TICs that is triggered by AKT1 deficiency is also associated with a loss of the stem cell/mesenchymal phenotype and a recovery of epithelial-like markers. Finally, we define downstream effectors that are responsible for controlling the CSC-phenotype, such as FoxO-Bim, and the death of these cells in the absence of AKT1. In summary, these data closely link the maintenance of the stem cell-like phenotype and the survival of these cells to the AKT-FoxO-Bim pathway.

Enteropathogenic Escherichia coli and vaccinia virus do not require the family of WASP-interacting proteins for pathogen-induced actin assembly.

The human pathogens enteropathogenic Escherichia coli (EPEC) and vaccinia virus trigger actin assembly in host cells by activating the host adaptor Nck and the actin nucleation promoter neural Wiskott-Aldrich syndrome protein (N-WASP). EPEC translocates effector molecules into host cells via type III secretion, and the interaction between the translocated intimin receptor (Tir) and the bacterial membrane protein intimin stimulates Nck and N-WASP recruitment, leading to the formation of actin pedestals beneath adherent bacteria. Vaccinia virus also recruits Nck and N-WASP to generate actin tails that promote cell-to-cell spread of the virus. In addition to Nck and N-WASP, WASP-interacting protein (WIP) localizes to vaccinia virus tails, and inhibition of actin tail formation upon ectopic expression of WIP mutants led to the suggestion that WIP is required for this process. Similar studies of WIP mutants, however, did not affect the ability of EPEC to form actin pedestals, arguing against an essential role for WIP in EPEC-induced actin assembly. In this study, we demonstrate that Nck and N-WASP are normally recruited by vaccinia virus and EPEC in the absence of WIP, and neither WIP nor the WIP family members CR16 and WIRE/WICH are essential for pathogen induced actin assembly. In addition, although Nck binds EPEC Tir directly, N-WASP is required for its localization during pedestal formation. Overall, these data highlight similar pathogenic strategies shared by EPEC and vaccinia virus by demonstrating a requirement for both Nck and N-WASP, but not WIP or WIP family members in pathogen-induced actin assembly.

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 regulates signaling via the high affinity receptor for immunoglobulin E in mast cells.

Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts.

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with beta3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)-/- OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP-/- OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP-/- OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

Crosstalk between WIP and Rho family GTPases.

Through actin-binding proteins such as the neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP-interacting protein (WIP), the Rho family GTPases RhoA, Rac1 and Cdc42 are major modulators of the cytoskeleton. (N-)WASP and WIP control Rho GTPase activity in various cell types, either by direct WIP/(N-)WASP/Cdc42 or potential WIP/RhoA binding, or through secondary links that regulate GTPase distribution and/or transcription levels. WIP helps to regulate filopodium generation and participates in the Rac1-mediated ruffle formation that determines cell motility. In neurons, lack of WIP increases dendritic spine size and filamentous actin content in a RhoA-dependent manner. In contrast, WIP deficiency in an adenocarcinoma cell line significantly reduces RhoA levels. These data support a role for WIP in the GTPase-mediated regulation of numerous actin-related cell functions; we discuss the possibility that this WIP effect is linked to cell proliferative status.

WIP regulates the stability and localization of WASP to podosomes in migrating dendritic cells.

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), we examined the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs). Our 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. We 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, our 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.

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-ß. 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.

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.

Role of Akt Isoforms Controlling Cancer Stem Cell Survival, Phenotype and Self-Renewal.

The cancer stem cell (CSC) hypothesis suggests that tumours are maintained by a subpopulation of cells with stem cell properties. Although the existence of CSCs was initially described in human leukaemia, less evidence exists for CSCs in solid tumours. Recently, a CD133+ cell subpopulation was isolated from human brain tumoursexhibiting stem cell properties in vitro as well as the capacity to initiate tumours in vivo. In the present work, we try to summarize the data showing that some elements of the Phosphoinositide 3-kinase Class I (PI3K)/ Thymoma viral oncogene protein kinase (Akt) pathway, such the activity of PI3K Class I or Akt2, are necessary to maintain the CSC-like phenotype as well as survival of CSCs (also denoted as tumour-initiating cells (TICs)). Our data and other laboratory data permit a working hypothesis in which each Akt isoform plays an important and specific role in CSC/TIC growth, self-renewal, maintaining survival, and epithelial-mesenchymal transition (EMT) phenotype, not only in breast cancer, but also in glioma. We suggest that a more complete understanding is needed of the possible roles of isoforms in human tumours (iso-signalling determination). Thus, a comprehensive analysis of how hierarchical signalling is assembled during oncogenesis, how cancer landmarks are interconnected to favour CSC and tumour growth, and how some protein isoforms play a specific role in CSCs to ensure that survival and proliferation must be done in order to propose/generate new therapeutic approaches (alone or in combination with existing ones) to use against cancer.

A role for WASP Interacting Protein, WIP, in fibroblast adhesion, spreading and migration.

The WASP (Wiskott Aldrich Syndrome Protein) Interacting Protein, WIP, regulates actin polymerization and the formation of actin-rich structures such as filopodia and lamellipodia, each of which is involved in cellular adhesion, spreading and migration. To define the role for WIP in these activities, we analysed cell adhesion and spreading as well as the redistribution of polymerised actin and paxillin that occurred when fibroblasts were plated onto different substrata. We compared the effect of WIP overexpression (gain of function) with that of WIP deficiency (loss of function) on these parameters. WIP-overexpression delayed cellular adhesion and spreading, an effect that could be compensated for by exposure to Y-27632, a well characterized ROCK (Rho kinase) inhibitor. WIP overexpression augmented the phosphorylation of Erk and JNK induced by binding to fibronectin, suggesting that WIP participates in signal transduction pathways initiated by integrin engagement. Conversely, WIP deficiency accelerated fibroblast adhesion to plastic and led to the formation of enlarged focal adhesions. The influence of WIP on fibroblast migration was measured by scratch assay. WIP-overexpression reduced migration while WIP-deficiency increased it, suggesting that WIP acts as a negative regulator of fibroblast migration. Together, these findings suggest a novel role for WIP in fibroblast adhesion, spreading and migration.

WIP: a multifunctional protein involved in actin cytoskeleton regulation.

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

WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion.

Cancer cells form actin-rich degradative protrusions (invasive pseudopods and invadopodia), which allows their efficient dispersal during metastasis. Using biochemical and advanced imaging approaches, we demonstrate that the N-WASP-interactors WIP and WICH/WIRE play non-redundant roles in cancer cell invasion. WIP interacts with N-WASP and cortactin and is essential for invadopodium assembly, whereas WICH/WIRE regulates N-WASP activation to control invadopodium maturation and degradative activity. Our data also show that Nck interaction with WIP and WICH/WIRE modulates invadopodium maturation; changes in WIP and WICH/WIRE levels induce differential distribution of Nck. We show that WIP can replace WICH/WIRE functions and that elevated WIP levels correlate with high invasiveness. These findings identify a role for WICH/WIRE in invasiveness and highlight WIP as a hub for signaling molecule recruitment during invadopodium generation and cancer progression, as well as a potential diagnostic biomarker and an optimal target for therapeutic approaches.

Neuritic complexity of hippocampal neurons depends on WIP-mediated mTORC1 and Abl family kinases activities.

INTRODUCTION: Neuronal morphogenesis is governed mainly by two interconnected processes, cytoskeletal reorganization, and signal transduction. The actin-binding molecule WIP (Wiskott-Aldrich syndrome protein [WASP]-interacting protein) was identified as a negative regulator of neuritogenesis. Although WIP controls activity of the actin-nucleation-promoting factor neural WASP (N-WASP) during neuritic differentiation, its implication in signal transduction remains unknown. METHODS: Using primary neurons from WIP-deficient and wild-type mice we did an immunofluorescence, morphometric, and biochemical analysis of the signaling modified by WIP deficiency. RESULTS: Here, we describe the WIP contribution to the regulation of neuritic elaboration and ramification through modification in phosphorylation levels of several kinases that participate in the mammalian target of rapamycin complex 1 (mTORC1)-p70S6K (phosphoprotein 70 ribosomal protein S6 kinase, S6K) intracellular signaling pathway. WIP deficiency induces an increase in the number of neuritic bifurcations and filopodial protrusions in primary embryonic neurons. This phenotype is not due to modifications in the activity of the phosphoinositide 3 kinase (PI3K)-Akt pathway, but to reduced phosphorylation of the S6K residues Ser(411) and Thr(389). The resulting decrease in kinase activity leads to reduced S6 phosphorylation in the absence of WIP. Incubation of control neurons with pharmacological inhibitors of mTORC1 or Abl, two S6K regulators, conferred a morphology resembling that of WIP-deficient neurons. Moreover, the preferential co-distribution of phospho-S6K with polymerized actin is altered in WIP-deficient neurons. CONCLUSION: These experiments identify WIP as a member of a signaling cascade comprised of Abl family kinases, mTORC1 and S6K, which regulates neuron development and specifically, neuritic branching and complexity. Thus, we postulated a new role for WIP protein.

WIP is necessary for matrix invasion by breast cancer cells.

Actin filament assembly and reorganisation during cell migration and invasion into extracellular matrices is a well-documented phenomenon. Among actin-binding proteins regulating its polymerisation, the members of the WASP (Wiskott Aldrich Syndrome Protein) family are generally thought to play the most significant role in supporting cell invasiveness. In situ, cytosolic N-WASP (neural WASP) is associated with a partner protein termed WIP (WASP Interacting Protein) that is bound to the N-terminal domain of N-WASP. Despite much effort, rather little is known about the role of WIP in regulating N-WASP and consequent actin-filament assembly. Even less is known about the function of WIP within the specialised cell adhesion and attachment structures known as podosomes and invadopodia. In particular, whilst the interaction of WIP with known participants in the development and maturation of invadopodia such as N-WASP, the Arp2/3 complex and cortactin has been described, little is known concerning the direct contribution of WIP to invadopodia and its potential role as a regulator of cancer cell invasion. In this report, we use 2D and 3D culture systems to describe the role played by WIP in modulating the morphology and invasiveness of metastatic breast cancer cells in vitro, as well as its effect on the process of mesenchymal-epithelial transition (MET) seen in these cells. We demonstrate that WIP is necessary for invadopodium formation and matrix degradation by basal breast cancer cells, but not sufficient to induce invasiveness in luminal cells.

Integrin linked kinase (ILK) regulates podosome maturation and stability in dendritic cells.

Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.

Phosphoinositide 3-kinase p85beta regulates invadopodium formation.

The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85ß, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85ß might facilitate cell invasion. We show that p85ß localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85ß induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85ß lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85ß function in invadopodium-like formation, p85ß levels increased in metastatic melanoma and p85ß depletion reduced invadopodium formation and invasion. These results show that p85ß enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85ß levels.