Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold.
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WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl. (+info)
Profilin enhances Cdc42-induced nucleation of actin polymerization.
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We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline. Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex. (+info)
Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate.
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Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from Cdc42 to the nucleation of actin filaments by Arp2/3 complex. Although full-length N-WASP is a weak activator of Arp2/3 complex, its activity can be enhanced by upstream regulators such as Cdc42 and PI(4,5)P(2). We dissected this activation reaction and found that the previously described physical interaction between the NH(2)-terminal domain and the COOH-terminal effector domain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH(2)- and COOH termini must be intramolecular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than through the WASp homology domain 1. Like Cdc42, PI(4,5)P(2) reduces the affinity between the NH(2)- and COOH termini of the molecule. The use of a mutant N-WASP molecule lacking this basic stretch allowed us to delineate a signaling pathway in Xenopus extracts leading from PI(4, 5)P(2) to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P(2) serves two functions: first, as an activator of N-WASP; and second, as an indirect activator of Cdc42. (+info)
Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex.
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We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 microM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP(2)) micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP(2) and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42 was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 microM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction. (+info)
Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation.
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Unrelated cord blood (UCB) is being used as a source of alternative hematopoietic stem cells for transplantation with increasing frequency. From November 1994 to February 1999, 30 UCB transplant procedures were performed for both malignant and nonmalignant diseases in 27 children, aged 0.4 to 17.1 years. Patients received either HLA-matched (n = 3) or 1- or 2-antigen-mismatched (n = 27) UCB following 1 of 2 standardized preparative and graft-versus-host disease regimens (hyperfractionated total body irradiation, cyclophosphamide, and antithymocyte globulin [ATG] with cyclosporine A and methotrexate; or busulfan, melphalan, and ATG with cyclosporine A and prednisone). The median time to neutrophil and platelet engraftment was 27 days (12-60 days) and 75 days (33-158 days) posttransplantation, respectively. No correlation was noted between neutrophil and platelet engraftment and nucleated cells per kilogram, CD34(+) cells per kilogram infused, or cytomegalovirus status of recipient. The cumulative probability of acute grade 2 or greater graft-versus-host disease (GVHD) was 37.2%, and of grade 3 or greater GVHD was 8.8%. No patients developed chronic GVHD. CD4, CD19, and natural killer cell recovery was achieved at a median of 12, 6, and 2 months, respectively. CD8 recovery was delayed at a median of 9 months. Normal mitogen response was achieved at 6 to 9 months. The probability of survival, disease-free survival, and event-free survival at 1 year was 52.3% (34.1%-70.5%), 54.7% (34.5%-74.9 %) and 49.6% (29.9%-69.4%), respectively. This series of 30 UCB transplants suggests that although CD8 cell recovery is delayed, the pattern of immune reconstitution with UCB is similar to that reported for other stem cell sources. (Blood. 2000;96:2703-2711) (+info)
Randomized trial of transfer factor treatment of human warts.
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Dialysed transfer factor, prepared from the leucocytes of a donor whose warts had undergone recent spontaneous regression, was used in the treatment of a child with the Wiskott--Aldrich syndrome. The child then had a spontaneous regression at multiple warty areas. A similar relationship was seen in four otherwise healthy patients in a pilot study. A randomized double-blind study of thirty patients failed to confirm a causal relationship between the transfer factor therapy (equivalent to 2-1 X 10(8) leucocytes) and wart regressions. The need for randomized trials of transfer factor therapy for diseases with a variable natural history is emphasized. (+info)
Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells.
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Stimulation of T cells via the antigen and costimulatory receptors leads to the organization of a supramolecular activation cluster called the immune synapse. We report that loss of the molecular adaptor Cbl-b in T cells frees antigen receptor-triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimulation. Introduction of the cbl-b mutation into a vav1-/- background relieved the functional defects of vav1-/- T cells and caused spontaneous autoimmunity. Wiscott Aldrich Syndrome protein (WASP) was found to be essential for deregulated proliferation and membrane receptor reorganization of cbl-b mutant T cells. Antigen receptor-triggered Ca2+ mobilization, cytokine production, and receptor clustering can be genetically uncoupled in cbl-b mutant T cells. Thus, Cbl-b functions as a negative regulator of receptor clustering and raft aggregation in T cells. (+info)
The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis.
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In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell-derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1-induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1-responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus-TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1-induced chemotaxis of T cells. (+info)