Phosphorylation Of Wiskott-Aldrich Syndrome Protein Research Articles

LINC00869 Promotes Hepatocellular Carcinoma Metastasis via Protrusion Formation.

LncRNA LINC00869 regulates the activity of CDC42-WASP pathway and positively affects protrusion formation in HCC cells, which expands the current understanding of lncRNA functions as well as gives a better understanding of carcinogenesis.

NPM-ALK phosphorylates WASp Y102 and contributes to oncogenesis of anaplastic large cell lymphoma.

Mechanisms by which NPM-ALK signaling regulates cell migration, invasion and contributes to the oncogenesis of anaplastic large cell lymphoma (ALCL) are not completely understood. In an attempt to identify novel actin signaling pathways regulated by NPM-ALK, a comprehensive phosphoproteome analysis of ALCL cell lines was performed in the presence or absence of NPM-ALK activity. Numerous phosphoproteins involved in actin dynamics including Wiskott-Aldrich syndrome protein (WASp) were regulated by NPM-ALK. Network analysis revealed that WASp is a central component of the NPM-ALK-dependent actin signaling pathway. Here we show that NPM-ALK phosphorylates WASp at its known activation site (Y290) as well as at a novel residue (Y102). Phosphorylation of WASp at Y102 negatively regulates its interaction with Wiskott-Aldrich interacting protein and decreases its protein stability. Phosphorylation of WASp at Y102 enhances anchorage-independent growth and tumor growth in an in vivo xenograft model and enhances invasive properties of ALCL. We show that knock-down of WASp or expression of Y102F mutant of WASp decreases colony formation and in vivo tumor growth. Our results show that WASp is a novel substrate of ALK and has a critical role in regulating invasiveness and oncogenesis of ALCL.

WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts.

The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti-ICAM-1 and anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability.

Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages

Wiskott–Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1β and phosphorylation of inhibitor of κB α/β (IKKα/β) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages.

Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) by Hck Regulates Macrophage Function

We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck(-/-) bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck(-/-) BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP(-/-) BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.

Wiskott–Aldrich Syndrome causing mutation, Pro373Ser restricts conformational changes essential for WASP activity in T-cells

Wiskott–Aldrich Syndrome (WAS) is caused by mutations in Wiskott-Aldrich Syndrome Protein (WASP) and majority of the mutations are found in the WASP Homology 1 (WH1) domain which mediates interaction with WIP (WASP Interacting Protein), a WASP chaperone. Two point mutations together in the proline rich region (PRR) domain of WASP (S339Y/P373S) have been reported to cause WAS however the molecular defect has not been characterized. Expression of these mutants separately (WASPRS339Y, WASPRP373S) or together (WASPRSP/YS) did not rescue the chemotaxis defect or membrane projection defect of JurkatWKD T-cells (WASP knockdown). This is not due to the inability of WASP-PRR mutants to form functional WASP–WIP complex in growth rescue experiments in las17Δ yeast strain. Expression of WASPRS339Y but not WASPRP373S or WASPRSP/YS rescued the IL-2 expression defect of JurkatWKD T-cells, suggesting that Pro373Ser mutation alone is sufficient to inhibit WASP functions in T-cell activation. The diffused localization of WASP-PRR mutants in activated Jurkat T-cells suggests that Ser339 and Pro373 are critical for WASP localization. WASP-PRR mutations either together or individually did not abolish interaction of WASP with sixteen WASP binding proteins including Hck, however they caused reduction in Hck mediated tyrosine phosphorylation of WASP which is critical for WASP activity. The auto-inhibitory conformation of WASPP373S mutant was not relieved by the binding of Toca-1 or Nck1. Thus, our results suggest that Pro373Ser mutation reduces Tyr291 phosphorylation and prevents conformational changes required for WASP activity in chemotaxis and T-cell activation. Thus Pro3373Ser is probably responsible for all the defects associated with WAS in the patients.

Wiskott-Aldrich Syndrome Protein (WASp) Controls the Delivery of Platelet Transforming Growth Factor-β1

Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp(-/-)) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10-15% released after 30-60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp(-/-) platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp(-/-) platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.

Ubiquitylation-Dependent Negative Regulation of WASp Is Essential for Actin Cytoskeleton Dynamics

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin dynamics during cell motility and adhesion, and mutations in its gene are responsible for Wiskott-Aldrich syndrome (WAS). Here, we demonstrate that WASp is ubiquitylated following T-cell antigen receptor (TCR) activation. WASp phosphorylation at tyrosine 291 results in recruitment of the E3 ligase Cbl-b, which, together with c-Cbl, carries out WASp ubiquitylation. Lysine residues 76 and 81, located at the WASp WH1 domain, which contains the vast majority of WASp gene mutations, serve as the ubiquitylation sites. Disruption of WASp ubiquitylation causes WASp accumulation and alters actin dynamics and the formation of actin-dependent structures. Our data suggest that regulated degradation of activated WASp might be an efficient strategy by which the duration and localization of actin rearrangement and the intensity of T-cell activation are controlled.

Single-chain variable fragment intrabody impairs LPS-induced inflammatory responses by interfering with the interaction between the WASP N-terminal domain and Btk in macrophages

Wiskott–Aldrich syndrome protein (WASP) plays important roles in both acquired and innate immune responses. We recently uncovered that the WASP N-terminal domain specifically binds the Src homology (SH) 3 domain of Bruton’s tyrosine kinase (Btk) in macrophages. Over-expression of the WASP N-terminal domain impairs LPS-induced inflammatory responses. To evaluate the significance of this interaction in LPS signaling, we established bone marrow-derived macrophage (BMDM) cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) intrabody. Anti-WASP scFv specifically bound endogenous WASP and inhibited its specific binding to the SH3 domain of Btk in the Tg BMDMs. Tyrosine phosphorylation in WASP was inhibited after LPS stimulation. As a result, TNF-α, IL-6, and IL-1β gene transcription and NF-κB phosphorylation were impaired. These observations strongly suggest that the phosphorylation of WASP by Btk plays a pivotal role in transducing the LPS signaling pathway in macrophages.

Pivotal roles of both Wiskott-Aldrich syndrome protein (WASP) and N-WASP in the activation and attenuation of the B cell receptor (P1093) (42.1)

Abstract Lymphocytes express both Wiskott-Aldrich syndrome protein (WASP) and N-WASP. WASP deficiency, the cause of WAS, generates more severe defects in T-cells than B cells, suggesting a strong compensatory role of N-WASP in B cells. Using knockout mouse models, this study shows that WASP knockout (WKO) reduces B cell spreading, B cell receptor (BCR) aggregation, tyrosine phosphorylation, Btk phosphorylation and F-actin accumulation at the B cell surface induced by membrane-associated antigen, and WASP/N-WASP double knockout nearly abolished these activities. However, in B cells with N-WASP conditional knockout (cNKO), B cell spreading and F-actin accumulation are enhanced, while the merge of BCR aggregates and attenuation of tyrosine and Btk phosphorylation are inhibited. Additionally, cNKO has a much stronger inhibitory effect on BCR endocytosis than WKO. In contrast to Btk-dependent activation of WASP, N-WASP phosphorylation is upregulated by Btk-deficiency but downregulated by SHIP KO. Furthermore, the phosphorylation of WASP and N-WASP is inversely regulated by each other. These results reveal that the combined function of WASP and N-WASP is required for the optimal activation of the BCR and its signaling attenuation, but these two proteins function distinctively: WASP and N-WASP play dominant roles in the initiation and attenuation of BCR activation respectively. Thus, the integrated action of WASP and N-WASP provide a critical regulatory mechanism for BCR activation.

The Chemotactic Defect in Wiskott-Aldrich Syndrome Macrophages Is Due to the Reduced Persistence of Directional Protrusions

Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42 in vivo suggest that WASp plays a role in directional sensing, however, its precise role in macrophage chemotaxis is still unclear. Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp−/− bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 – elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1.

SGK1 Sensitivity of Platelet Migration

Recent observations pointed to the ability of platelets to migrate and thus to invade the inflamed vascular wall. Platelet migration could be stimulated by stromal cell-derived factor-1 (SDF-1), an effect dependent on phosphatidylinositide-3-kinase (PI3K) and paralleled by activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP). Migration is inhibited by vinculin, which is similarly regulated by phosphorylation. PI3K-sensitive kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored whether SGK1 modifies WASP and vinculin phosphorylation in murine platelets and participates in the regulation of platelet migration. Platelets were isolated from gene-targeted mice lacking SGK1 (sgk1^-/-) and from their wild type littermates (sgk1^+/+). Platelet migration stimulated with SDF-1 was significantly less pronounced in sgk1^-/-platelets than in sgk1^+/+ platelets. Moreover, SDF-1 significantly induced WASP phosphorylation, an effect again reduced in platelets lacking SGK1. Phosphorylation of vinculin was significantly enhanced in sgk1^-/-platelets and was significantly reduced following treatment of platelets with Ca²⁺ chelator BAPTA. Immunohistochemical analysis of in vivo experiments in intestinal vessels after vascular inflammation revealed that transmigration of platelets into inflamed vessel walls was significantly less pronounced in sgk1^-/-than in sgk1^+/+ mice. In conclusion, SGK1 is a powerful regulator of platelet migration.

Identification of Fyn as the binding partner for the WASP N-terminal domain in T cells

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling. In transgenic (Tg) mice, over-expression of the WASP N-terminal region (exons 1-5) including the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain and anti-WASP-EVH1 single-chain variable fragment (scFv) intracellular expressed antibodies (intrabodies) impairs IL-2 production in activated T cells. However, it largely remains unknown that how this domain transduces TCR signaling. Here, we demonstrate for the first time that the WASP N-terminal domain specifically associates with the Fyn SH3 domain; the interaction was uncovered by screening a λgt11 cDNA expression library obtained from the mouse T-cell line KKF. The interaction between Fyn and WASP was inhibited by over-expression of the WASP N-terminal domain and anti-WASP-EVH1 scFv intrabodies in gene-transfected NIH3T3 cells and T cells derived from these Tg mice. WASP-interacting protein binding to the EVH1 domain of WASP was also inhibited in these Tg mice T cells. Furthermore, tyrosine phosphorylation of WASP and nuclear translocation of nuclear factor of activated T cells following TCR stimulation was severely inhibited by over-expression of the WASP N-terminal domain. These observations strongly suggest that the WASP N-terminal domain plays a pivotal role in the TCR signaling cascade by binding to Fyn.

Syk Regulates Multiple Signaling Pathways Leading to CX3CL1 Chemotaxis in Macrophages

Several studies have clearly established the importance of the interaction between macrophages and CX3CL1 in the progression of disease. A previous study demonstrated that Syk was required for CX3CL1-mediated actin polymerization and chemotaxis. Here, we delineated the signaling cascade of Syk-mediated cell migration in response to CX3CL1. Inhibition of Syk in bone marrow-derived macrophages or reduction of Syk expression using siRNA in RAW/LR5 cells indicated that Syk was required for the activation of PI3K, Cdc42, and Rac1. Also, reduction in WASP or WAVE2 levels, common downstream effectors of Cdc42 or Rac1, resulted in impaired cell migration to CX3CL1. Syk indirectly regulated WASP tyrosine phosphorylation through Cdc42 activation. Altogether, our data identify that Syk mediated chemotaxis toward CX3CL1 by regulating both Rac1/WAVE2 and Cdc42/WASP pathways, whereas Src family kinases were required for proper WASP tyrosine phosphorylation.

Tyrosine Phosphorylation of WASP Promotes Calpain-Mediated Podosome Disassembly In Myeloid Cells.

AbstractAbstract 1498We have previously shown that a point mutation (Ile294Thr) in the Wiskott Aldrich Syndrome Protein (WASP) detected in a Wiskott Aldrich Syndrome (WAS) patient led to enhanced actin polymerising activity of WASP 1 and well as increased instability2. We also showed that macrophages from this patient displayed an increased number of actin based adhesion structures called podosomes. Additionally, podosomes in macrophages from this patient were extremely dynamic with a high rate of turnover. Based on these results we proposed that the active open conformation of WASP promotes actin polymerisation but it also induces podosome disassembly and adhesion turnover. Although the idea that the same open conformation of WASP leads to both assembly and disassembly of podosomes may be counterintuitive at first, it is possible that for termination of podosomes actin polymerisation and integrin recruitment have to be discontinued and the same constituents of growing podosomes such as active WASP may contribute to the disassembly process. We have also shown that the rapid turn-over of podosomes involves cleavage of WASP by the protease calpain, further supporting a role of WASP in both podosome formation and disassembly. However, the specific signalling mechanisms that make active WASP susceptible to cleavage by calpain leading to podosome disassembly remain unknown and need further clarification. Phosphorylation of WASP Y291 (human) or Y293 (mouse) promotes the open conformation of WASP and results in enhanced actin polymerisation. We now report that in myeloid cells, tyrosine phosphorylation of WASP negatively regulates the stability of podosomes, leading to their calpain-dependent disassembly. Additionally, we found that constitutive phosphorylation of WASP results in extensive degradation in a process that involves calpain. Our data also indicate that phosphorylation of WASP sustains the open/active conformation that promotes cleavage of WASP by calpain. Interestingly, phosphorylated WASP can bind to the WASP interacting protein (WIP) and other proteins that form a complex with WASP in podosomes such as Nck, cortactin. Taken together, our data indicate that in myeloid cells, tyrosine phosphorylation sustains the open conformation of WASP and it enhances its susceptibility to calpain-mediated cleavage preventing accumulation of actin filaments and integrin associated proteins in podosomes. This process facilitates podosome disassembly and cell translocation. These new findings support the key role of WASP as a protein that integrates actin polymerisation and cell adhesion required for mobilisation of myeloid cells during the immune response.Reference List1. Ancliff PJ, Blundell MP, Cory GO et al. Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia. Blood 2006;108:2182-2189.2. Moulding DA, Blundell MP, Spiller DG et al. Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia. J Exp. Med 2007;204:2213–2224. Disclosures:No relevant conflicts of interest to declare.

PI3 kinase-dependent stimulation of platelet migration by stromal cell-derived factor 1 (SDF-1)

Platelets have been regarded as static cells that do not move once they adhere to a matrix. The present study explored, whether platelets are able to migrate. In contrast to the current opinion, we found that platelets were mobile, able to migrate over a surface, and transmigrate through a transwell membrane and endothelium toward a source of stromal cell-derived factor 1 (SDF-1). Platelet migration was stimulated by SDF-1, which led to the downstream activation and phosphorylation of Wiskott-Aldrich syndrome protein. SDF-1 signaling and subsequent platelet migration could be inhibited by CXCR4-receptor blocker AMD3100, pertussis toxin, inhibition of phosphoinositol 3-kinase (PI3 kinase) with LY294002 or wortmannin, and disruption of actin polymerization with cytochalasin B. The potential of platelets to migrate in an SDF-1-mediated fashion may redefine the role of platelets in the pathophysiology of vascular inflammation, subsequent atherosclerotic degeneration, and vascular regeneration.

Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages

Podosomes, adhesion structures capable of matrix degradation, have been linked with the ability of cells to perform chemotaxis and invade tissues. Wiskott-Aldrich Syndrome protein (WASp), an effector of the RhoGTPase Cdc42 and a Src family kinase substrate, regulates macrophage podosome formation. In this study, we demonstrate that WASp is active in podosomes by using TIRF-FRET microscopy. Pharmacological and RNA interference approaches suggested that continuous WASp activity is required for podosome formation and function. Rescue experiments using point mutations demonstrate an absolute requirement for Cdc42 binding to WASp in podosome formation. Although tyrosine phosphorylation was not absolutely required for podosome formation, phosphorylation did regulate the rate of podosome nucleation and actin filament stability. Importantly, WASp tyrosine phosphorylation does not alter WASp activation, instead phosphorylation appears to be important for the restriction of WASp activity to podosomes. In addition, the matrix-degrading ability of cells requires WASp phosphorylation. Chemotactic responses to CSF-1 were also attenuated in the absence of endogenous WASp, which could not be rescued with either tyrosine mutation. These results suggest a more complex role for tyrosine phosphorylation than simply in the regulation of WASp activity, and suggest a link between podosome dynamics and macrophage migration.

Wiskott-Aldrich syndrome protein is an effector of Kit signaling

The pleiotropic receptor tyrosine kinase Kit can provide cytoskeletal signals that define cell shape, positioning, and migration, but the underlying mechanisms are less well understood. In this study, we provide evidence that Kit signals through Wiskott-Aldrich syndrome protein (WASP), the central hematopoietic actin nucleation-promoting factor and regulator of the cytoskeleton. Kit ligand (KL) stimulation resulted in transient tyrosine phosphorylation of WASP, as well as interacting proteins WASP-interacting protein and Arp2/3. KL-induced filopodia in bone marrow-derived mast cells (BMMCs) were significantly decreased in number and size in the absence of WASP. KL-dependent regulation of intracellular Ca(2+) levels was aberrant in WASP-deficient BMMCs. When BMMCs were derived from WASP-heterozygous female mice using KL as a growth factor, the cultures eventually developed from a mixture of WASP-positive and -negative populations into a homogenous WASP-positive culture derived from the WASP-positive progenitors. Thus, WASP expression conferred a selective advantage to the development of Kit-dependent hematopoiesis consistent with the selective advantage of WASP-positive hematopoietic cells observed in WAS-heterozygous female humans. Finally, KL-mediated gene expression in wild-type and WASP-deficient BMMCs was compared and revealed that approximately 30% of all Kit-induced changes were WASP dependent. The results indicate that Kit signaling through WASP is necessary for normal Kit-mediated filopodia formation, cell survival, and gene expression, and provide new insight into the mechanism in which WASP exerts a strong selective pressure in hematopoiesis.

Phosphorylation of WASp is a key regulator of activity and stability in vivo

The Wiskott-Aldrich syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. Covalent modification of a conserved tyrosine by phosphorylation has emerged as an important potential determinant of activity, although the physiological significance remains uncertain. In a murine knockin model, mutation resulting in inability to phosphorylate Y293 (Y293F) mimicked many features of complete WASp-deficiency. Although a phosphomimicking mutant Y293E conferred enhanced actin-polymerization, the cellular phenotype was similar due to functional dysregulation. Furthermore, steady-state levels of Y293E-WASp were markedly reduced compared to wild-type WASp and Y293F-WASp, although partially recoverable by treatment of cells with proteasome inhibitors. Consequently, tyrosine phosphorylation plays a critical role in normal activation of WASp in vivo, and is indispensible for multiple tasks including proliferation, phagocytosis, chemotaxis, and assembly of adhesion structures. Furthermore, it may target WASp for proteasome-mediated degradation, thereby providing a default mechanism for self-limiting stimulation of the Arp2/3 complex.

Cdc42 Regulates FcγReceptor-mediated Phagocytosis through the Activation and Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) and Neural-WASP

Cdc42 is a key regulator of the actin cytoskeleton and activator of Wiskott-Aldrich syndrome protein (WASP). Although several studies have separately demonstrated the requirement for both Cdc42 and WASP in Fc(gamma) receptor (Fc(gamma)R)-mediated phagocytosis, their precise roles in the signal cascade leading to engulfment are still unclear. Reduction of endogenous Cdc42 expression by using RNA-mediated interference (short hairpin RNA [shRNA]) severely impaired the phagocytic capacity of RAW/LR5 macrophages, due to defects in phagocytic cup formation, actin assembly, and pseudopod extension. Addition of wiskostatin, a WASP/neural-WASP (N-WASP) inhibitor showed extensive inhibition of phagocytosis, actin assembly, and cell extension identical to the phenotype seen upon reduction of Cdc42 expression. However, using WASP-deficient bone marrow-derived macrophages or shRNA of WASP or N-WASP indicated a requirement for both WASP and N-WASP in phagocytosis. Cdc42 was necessary for WASP/N-WASP activation, as determined using a conformation-sensitive antibody against WASP/N-WASP and partial restoration of phagocytosis in Cdc42 reduced cells by expression of a constitutively activated WASP. In addition, Cdc42 was required for proper WASP tyrosine phosphorylation, which was also necessary for phagocytosis. These results indicate that Cdc42 is essential for the activation of WASP and N-WASP, leading to actin assembly and phagocytic cup formation by macrophages during Fc(gamma)R-mediated phagocytosis.