title: Identification of another actin-related protein (Arp) 2/3 complex binding site in neural Wiskott-Aldrich syndrome protein (N-WASP) that complements actin polymerization induced by the Arp2/3 complex activating (VCA) domain of N-WASP.
abstract: Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.
authors: Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 1088639, Japan.
authors: S
authors: S
authors: Suetsugu
authors: H
authors: H
authors: Miki
authors: T
authors: T
authors: Takenawa
mesh_terms: D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003598:Cytoskeletal Proteins; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D014923:Wiskott-Aldrich Syndrome / Q000235:genetics; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
chemical_list: C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal