Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate.
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.
Rohatgi
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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Kirschner
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D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D003598:Cytoskeletal Proteins; D019204:GTP-Binding Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D014923:Wiskott-Aldrich Syndrome / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D014981:Xenopus; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D009419:Nerve Tissue Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011108:Polymers; C495770:Wasl protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein