title: Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities.
abstract: BACKGROUND
  Assembly and organization of actin filaments are required for many cellular processes, including locomotion and division. In many cases, actin assembly is initiated when proteins of the WASP/Scar family respond to signals from Rho family G proteins and stimulate the actin-nucleating activity of the Arp2/3 complex. Two questions of fundamental importance raised in the study of actin dynamics concern the molecular mechanism of Arp2/3-dependent actin nucleation and how different signaling pathways that activate the same Arp2/3 complex produce actin networks with different three-dimensional architectures?
  
  
  RESULTS
  We directly compared the activity of the Arp2/3 complex in the presence of saturating concentrations of the minimal Arp2/3-activating domains of WASP, N-WASP, and Scar1 and found that each induces unique kinetics of actin assembly. In cell extracts, N-WASP induces rapid actin polymerization, while Scar1 fails to induce detectable polymerization. Using purified proteins, Scar1 induces the slowest rate of nucleation. WASP activity is 16-fold higher, and N-WASP activity is 70-fold higher. The data for all activators fit a mathematical model in which one activated Arp2/3 complex, one actin monomer, and an actin filament combine into a preactivation complex which then undergoes a first-order activation step to become a nucleus. The differences between Scar and N-WASP activity are explained by differences in the rate constants for the activation step. Changing the number of actin binding sites on a WASP family protein, either by removing a WH2 domain from N-WASP or by adding WH2 domains to Scar1, has no significant effect on nucleation activity. The addition of a three amino acid insertion found in the C-terminal acidic domains of WASP and N-WASP, however, increases the activity of Scar1 by more than 20-fold. Using chemical crosslinking assays, we determined that both N-WASP and Scar1 induce a conformational change in the Arp2/3 complex but crosslink with different efficiencies to the small molecular weight subunits p18 and p14.
  
  
  CONCLUSIONS
  The WA domains of N-WASP, WASP, and Scar1 bind actin and Arp2/3 with nearly identical affinities but stimulate rates of actin nucleation that vary by almost 100-fold. The differences in nucleation rate are caused by differences in the number of acidic amino acids at the C terminus, so each protein is tuned to produce a different rate of actin filament formation. Arp2/3, therefore, is not regulated by a simple on-off switch. Precise tuning of the filament formation rate may help determine the architecture of actin networks produced by different nucleation-promoting factors.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California-San Francisco, 94143, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zalevsky
authors.forename: L
authors.initials: L
authors.lastname: Lempert
authors.forename: H
authors.initials: H
authors.lastname: Kranitz
authors.forename: R D
authors.initials: RD
authors.lastname: Mullins
mesh_terms: D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D001704:Biopolymers; D003598:Cytoskeletal Proteins; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000502:physiology*; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family; 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; D001704:Biopolymers; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; C495767:WAS protein, human; C495771:WASL protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal