Abstract
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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins / metabolism*
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Animals
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Antibodies
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Cattle
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Cytoskeletal Proteins*
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GTP-Binding Proteins / metabolism
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Humans
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Lipid Metabolism
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Membrane Proteins / metabolism
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Microscopy, Fluorescence
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Muscle, Skeletal / metabolism
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Nerve Tissue Proteins / immunology
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Nerve Tissue Proteins / isolation & purification
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Nerve Tissue Proteins / metabolism*
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Neutrophils / metabolism
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Phosphatidylinositol 4,5-Diphosphate / metabolism*
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Protein Prenylation
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Rabbits
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Thymus Gland / metabolism
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Wiskott-Aldrich Syndrome / metabolism*
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Wiskott-Aldrich Syndrome Protein, Neuronal
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cdc42 GTP-Binding Protein / metabolism*
Substances
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ACTR2 protein, human
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ACTR3 protein, human
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins
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Antibodies
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Cytoskeletal Proteins
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Membrane Proteins
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Nerve Tissue Proteins
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Phosphatidylinositol 4,5-Diphosphate
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WASL protein, human
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Wiskott-Aldrich Syndrome Protein, Neuronal
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GTP-Binding Proteins
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cdc42 GTP-Binding Protein