title: Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2.
abstract: The Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP) are key players in regulating actin cytoskeleton via the Arp2/3 complex. It has been widely reported that the WASP proteins are activated by Rho family small GTPase Cdc42 and that Rac1 acts through SCAR/WAVE proteins. However, a systematic study of the specificity of different GTPases for different Arp2/3 activators has not been conducted. In this study, we have expressed, purified, and characterized completely soluble, highly active, and autoinhibited full-length human WASP and N-WASP from mammalian cells. We show a novel N-WASP activation by Rho family small GTPase Rac1. This GTPase exclusively stimulates N-WASP and has no effects on WASP. Rac1 is a significantly more potent N-WASP activator than Cdc42. In contrast, Cdc42 is a more effective activator of WASP than N-WASP. Lipid vesicles containing PIP2 significantly improve actin nucleation by the Arp2/3 complex and N-WASP in the presence of Rac1 or Cdc42. PIP2 vesicles have no effect on WASP activity alone. Moreover, the inhibition of WASP-stimulated actin nucleation in the presence of Cdc42 and PIP2 vesicles has been observed. We found that adaptor proteins Nck1 or Nck2 are the most potent WASP and N-WASP activators with distinct effects on the WASP family members. Our in vitro data demonstrates differential regulation of full-length WASP and N-WASP by cellular activators that highlights fundamental differences of response at the protein-protein level.
authors.affiliation: Cytokinetics Inc., 280 East Grand Avenue, South San Francisco, California 94080, USA. ntomasevic@cytokinetics.com
authors.forename: Nenad
authors.initials: N
authors.lastname: Tomasevic
authors.forename: Zhiheng
authors.initials: Z
authors.lastname: Jia
authors.forename: Alan
authors.initials: A
authors.lastname: Russell
authors.forename: Toby
authors.initials: T
authors.lastname: Fujii
authors.forename: James J
authors.initials: JJ
authors.lastname: Hartman
authors.forename: Sheila
authors.initials: S
authors.lastname: Clancy
authors.forename: Manping
authors.initials: M
authors.lastname: Wang
authors.forename: Christophe
authors.initials: C
authors.lastname: Beraud
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Wood
authors.forename: Roman
authors.initials: R
authors.lastname: Sakowicz
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000502:physiology*; D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D006801:Humans; D015513:Oncogene Proteins / Q000502:physiology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D051316:Wiskott-Aldrich Syndrome Protein / Q000502:physiology*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000502:physiology*; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*; D020830:rac1 GTP-Binding Protein / Q000502:physiology*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D051316:Wiskott-Aldrich Syndrome Protein; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein