SLP-76 coordinates Nck-dependent Wiskott-Aldrich syndrome protein recruitment with Vav-1/Cdc42-dependent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact site

We have shown previously that Wiskott-Aldrich syndrome protein (WASP) activation at the site of T cell-APC interaction is a two-step process, with recruitment dependent on the proline-rich domain and activation dependent on binding of Cdc42-GTP to the GTPase binding domain. Here, we show that WASP recruitment occurs through binding to the C-terminal Src homology 3 domain of Nck. In contrast, WASP activation requires Vav-1. In Vav-1-deficient T cells, WASP recruitment proceeds normally, but localized activation of Cdc42 and WASP is disrupted. The recruitment and activation of WASP are coordinated by tyrosine-phosphorylated Src homology 2 domain-containing leukocyte protein of 76 kDa, which functions as a scaffold, bringing Nck and WASP into proximity with Vav-1 and Cdc42-GTP. Taken together, these findings reconstruct the signaling pathway leading from TCR ligation to localized WASP activation.     

Wiskott-Aldrich syndrome protein induces actin clustering without direct binding to Cdc42.

WASP (Wiskott-Aldrich syndrome protein) was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is(are) important for the function. Here we present for the first time the results of detailed analyses on the domain-function relationship of WASP. First, the C-terminal verprolin-cofilin-acidic domain was shown to be essential for the regulation of actin cytoskeleton. In addition, we found that the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clustered especially around the nucleus as wild type WASP without inducing actin clustering. Finally, we obtained the quite unexpected result that a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site.     

Protein-tyrosine kinase and GTPase signals cooperate to phosphorylate and activate Wiskott-Aldrich syndrome protein (WASP)/neuronal WASP

Protein-tyrosine kinases and Rho GTPases regulate many cellular processes, including the reorganization and dynamics of the actin cytoskeleton. The Wiskott-Aldrich syndrome protein (WASP) and its homolog neuronal WASP (N-WASP) are effectors of the Rho GTPase Cdc42 and provide a direct link between activated membrane receptors and the actin cytoskeleton. WASP and N-WASP are also regulated by a large number of other activators, including protein-tyrosine kinases, phosphoinositides, and Src homology 3-containing adaptor proteins, and can therefore serve as signal integrators inside cells. Here we show that Cdc42 and the Src family kinase Lck cooperate at two levels to enhance WASP activation. First, autoinhibition in N-WASP decreases the efficiency (kcat/Km) of phosphorylation and dephosphorylation of the GTPase binding domain by 30- and 40-fold, respectively, and this effect is largely reversed by Cdc42. Second, Cdc42 and the Src homology 3-Src homology 2 module of Lck cooperatively stimulate the activity of phosphorylated WASP, with coupling energy of approximately 2.4 kcal/mol between the two activators. These combined effects provide mechanisms for high specificity in WASP activation by coincident GTPase and kinase signals.     

Localization of the PAK1-, WASP-, and IQGAP1-specifying regions of Cdc42.

The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.     

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation

BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.     

Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).     

Investigation of the Interaction between Cdc42 and Its Effector TOCA1: HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES*

Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.     

E-cadherin-mediated cell-cell attachment activates Cdc42

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. Cdc42, a member of the Rho family of small GTPases, participates in cytoskeletal rearrangement and cell cycle progression. Recent evidence reveals that members of the Rho family modulate E-cadherin function. To further examine the role of Cdc42 in E-cadherin-mediated cell-cell adhesion, we developed an assay for active Cdc42 using the GTPase-binding domain of the Wiskott-Aldrich syndrome protein. Initiation of E-cadherin-mediated cell-cell attachment significantly increased in a time-dependent manner the amount of active Cdc42 in MCF-7 epithelial cell lysates. By contrast, Cdc42 activity was not increased under identical conditions in MCF-7 cells incubated with anti-E-cadherin antibodies nor in MDA-MB-231 (E-cadherin negative) epithelial cells. By fusing the Wiskott-Aldrich syndrome protein/GTPase-binding domain to a green fluorescent protein, activation of endogenous Cdc42 by E-cadherin was demonstrated in live cells. These data indicate that E-cadherin activates Cdc42, demonstrating bi-directional interactions between the Rho- and E-cadherin signaling pathways.     

Wiskott-Aldrich syndrome protein, WASP

Wiskott-Aldrich Syndrome protein (WASP) is the product of the gene mutated in children with Wiskott-Aldrich Syndrome (WAS). It is a predominantly cytoplasmic protein, expressed only in haematopoietic cells. It binds in vivo to the adaptor proteins Nck and Grb2, to the cytoplasmic protein-tyrosine kinase Fyn and to the small Rho-like GTPase Cdc42, which is required for formation of filopodia in fibroblasts and macrophages. WASP also interacts, directly or indirectly, with the actin cytoskeleton. Together with studies of a closely related, ubiquitously expressed protein named N-WASP, these findings suggest that WASP is a component of signalling pathways that control reorganisation of the actin cytoskeleton in haematopoietic cells in response to external stimuli. In support of this idea, haematopoietic cells from WAS patients show defects in cytoskeletal organisation that compromise their ability to polarise and to migrate in response to physiological stimuli. These defects could account for many of the clinical features of WAS. WAS is now a candidate for gene therapy based on the delivery of a wild-type WASP gene to autologous haematopoietic stem cells. In addition, recent studies of cell defects in WAS patients suggest that it may prove possible, in time, to rescue WAS cells using more conventional drug therapies.     

A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42

We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.     

Mutations that cause the Wiskott-Aldrich syndrome impair the interaction of Wiskott-Aldrich syndrome protein (WASP) with WASP interacting protein

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune deficiency, and a proclivity toward lymphoid malignancy. Lymphocytes of affected individuals show defects of activation, motility, and cytoskeletal structure. The disease gene encodes a 502-amino acid protein named the WAS protein (WASP). Studies have identified a number of important interactions that place WASP in a role of integrating signaling pathways with cytoskeletal function. We performed a two-hybrid screen to identify proteins interacting with WASP and cloned a proline-rich protein as a specific WASP interactor. Our clone of this protein, termed WASP interacting protein (WIP) by others, shows a difference in seven amino acid residues, compared with the previously published sequence revealing an additional profilin binding motif. Deletion mutant analysis reveals that WASP residues 101-151 are necessary for WASP-WIP interaction. Point mutant analyses in the two-hybrid system and in vitro show impairment of WASP-WIP interaction with three WASP missense mutants known to cause WAS. We conclude that impaired WASP-WIP interaction may contribute to WAS.     

Regulation of Wiskott-Aldrich syndrome protein and related molecules.

Wiskott-Aldrich syndrome protein (WASP), the gene mutated in the haematological disorder Wiskott-Aldrich syndrome, is the founding member of a family of conserved cytoskeletal regulators. WASP family proteins regulate actin dynamics through binding and activation of the Arp2/3 complex, which nucleates new actin filaments. Recently, a huge amount of information on WASPs has become available, both in terms of the function of individual domains, identification of their binding partners, and the unravelling of complex regulatory mechanisms. Together, these new findings place WASP-related molecules at the crossroads between distinct signalling pathways, which they integrate into coherent cytoskeletal responses.     

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.     

Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange.

The Cdc42 GTPase, a member of the Rho subfamily of Ras proteins, can signal to the cytoskeleton through its effector, the Wiskott-Aldrich syndrome protein (WASP), activation of which results in localized polymerization of new actin filaments. NMR structures of WASP peptide models in the Cdc42-bound and free states suggest that GTPase binding weakens autoinhibitory contacts between the GTPase binding domain (GBD) and the C-terminal actin regulatory (VCA) region of the protein. In the study presented here, amide hydrogen exchange has been used with NMR spectroscopy to directly examine destabilization of the autoinhibited GBD-VCA conformation caused by GTPase binding. A truncated protein, GBD-C, which models autoinhibited WASP, folds into a highly stable conformation with amide exchange protection factors of up to 3 x 10(6). A novel hydrogen exchange labeling-quench strategy, employing a high-affinity ligand to displace Cdc42 from WASP, was used to examine the amide exchange from the Cdc42-bound state of GBD-C. The GTPase increases exchange rates of the most protected amides by 50-500-fold, with destabilization reducing the differences in the protection of segments in the free state. The results confirm that Cdc42 facilitates the physical separation of the GBD from the VCA in a tethered molecule, indicating this process likely plays an important role in activation of full-length WASP by the GTPase. However, destabilization of GBD-C is not complete in the Cdc42 complex. The data indicate that partitioning of free energy between binding and activation may limit the extent to which GTPases can cause conformational change in effectors. This notion is consistent with the requirement of multiple input signals in order to achieve maximal activation in many effector molecules.     

Regulation of actin dynamics by WASP family proteins

Rapid reorganization of the actin cytoskeleton underlies morphological changes and motility of cells. WASP family proteins have received a great deal of attention as the signal-regulated molecular switches that initiate actin polymerization. The first member, WASP, was identified as the product of a gene of which dysfunction causes the human hereditary disease Wiskott-Aldrich syndrome. There are now five members in this protein family, namely WASP, N-WASP, WAVE/Scar1, 2, and 3. WASP and N-WASP have functional and physical associations with Cdc42, a Rho family small GTPase involved in filopodium formation. In contrast, there is evidence that links the WAVE/Scar proteins with another Rho family protein, Rac, which is a regulator of membrane ruffling. All WASP family members have a VCA domain at the C-terminus through which Arp2/3 complex is activated to nucleate actin polymerization. Analyses of model organisms have just begun to reveal unexpected functions of WASP family proteins in multicellular organisms.     

The human WASP-interacting protein, WIP, activates the cell polarity pathway in yeast.

WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.     

FBP11 regulates nuclear localization of N-WASP and inhibits N-WASP-dependent microspike formation

WASP family proteins are involved in cortical actin cytoskeleton reorganization. Neural Wiskott-Aldrich syndrome protein (N-WASP), a ubiquitously expressed WASP homologous protein, directly binds with Cdc42, activating Arp2/3 complex. In this study, we show that N-WASP-dependent microspike formation is inhibited by formin binding protein 11 (FBP11). Endogenous FBP11 localizes with nuclear-speckles, and co-localization of N-WASP and FBP11 was observed when they were co-expressed. Epidermal growth factor (EGF) induced actin-microspike formation in COS7 cells. However, transient expression of FBP11 suppressed N-WASP-dependent actin-microspike formation by trapping N-WASP in the nucleus. These results indicate that FBP11 regulates localization of N-WASP, thus negatively regulating the function of N-WASP in the cytoplasm.     

Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains.

In the second of a series of experiments designed to identify p47nck-Src homology 3 (SH3)-binding molecules, we report the cloning of SAKAP II (Src A box Nck-associated protein II) from an HL60 cDNA expression library. This molecule has been identified as a cDNA encoding the protein product of WASP, which is mutated in Wiskott-Aldrich syndrome patients. Studies in vivo and in vitro demonstrated a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antibodies recognized a protein of 66 kDa by Western blot (immunoblot) analysis. In vitro translation studies identified the 66-kDa protein as the protein product of WASP, and subcellular fractionation experiments showed that p66WASP is mainly present in the cytosol fraction, although significant amounts are also present in membrane and nuclear fractions. The main p47nck region implicated in the association with p66WASP was found to be the carboxy-terminal SH3 domain.     

A complex of Wiskott-Aldrich syndrome protein with mammalian verprolins plays an important role in monocyte chemotaxis

The Wiskott-Aldrich syndrome protein (WASP) is a product of the gene defective in an Xid disorder, Wiskott-Aldrich syndrome. WASP expression is limited to hemopoietic cells, and WASP regulates the actin cytoskeleton. It has been reported that monocytes/macrophages from WASP-deficient Wiskott-Aldrich syndrome patients are severely defective in chemotaxis, resulting in recurrent infection. However, the molecular basis of such chemotactic defects is not understood. Recently, the WASP N-terminal region was found to bind to the three mammalian verprolin homologs: WASP interacting protein (WIP); WIP and CR16 homologous protein (WICH)/WIP-related protein (WIRE); and CR16. Verprolin was originally found to play an important role in the regulation of actin cytoskeleton in yeast. We have shown that WASP, WIP, and WICH/WIRE are expressed predominantly in the human monocyte cell line THP-1 and that WIP and WICH/WIRE are involved in monocyte chemotaxis. When WASP binding to verprolins was blocked, chemotactic migration of monocytes was impaired in both THP-1 cells and primary human monocytes. Increased expression of WASP and WIP enhanced monocyte chemotaxis. Blocking WASP binding to verprolins impaired cell polarization but not actin polymerization. These results indicate that a complex of WASP with mammalian verprolins plays an important role in chemotaxis of monocytes. Our results suggest that WASP and mammalian verprolins function as a unit in monocyte chemotaxis and that the activity of this unit is critical to establish cell polarization. In addition, our results also indicate that the WASP-verprolin complex is involved in other functions such as podosome formation and phagocytosis.     

Specificity determinants on Cdc42 for binding its effector protein ACK

Cdc42 and Rac are highly homologous members of the Rho family of small G proteins that interact with several downstream effector proteins thereby causing cytoskeletal rearrangements, cell proliferation, and differentiation. While some effectors, such as the tyrosine kinase, ACK, and the scaffold protein, WASP, are unique to Cdc42, others, such as the serine-threonine kinase, PAK, are shared with Rac. Previous mutagenesis studies identified Val42 and Leu174 as residues that selectively affect binding of Cdc42 to ACK and WASP but not to PAK. However, it is unclear whether these discriminatory residues are sufficient determinants of specificity. In this study we sought to introduce "gain-of function" mutations into Rac to allow it to bind to ACK and WASP, thereby revealing all specificity determinants. Thirteen mutations were made changing Rac residues to those in Cdc42. Equilibrium binding constants of all mutant Rac proteins to ACK, WASP, and PAK were measured. A combination of seven mutations (S41A, A42V, N43T, D47G, N52T, W56F, and R174L) was determined to be necessary to change the binding affinity of Rac for ACK from negligible (K(d) < 1 microM) to a comparable affinity to Cdc42 (K(d) 25 nM). These mutations are not confined to interface residues. We interpret these data to indicate the importance of the structure of regions of the protein distinct from the contact residues. None of these mutant Rac proteins bound WASP with a similar affinity to Cdc42. Hence, residues as yet unidentified, outside the interface, must be necessary for binding WASP.