Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization.
The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent K(d) of 0.4 microM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp WA and <10 nM Arp2/3 complex, there is a 1:1 correspondence between the Arp2/3 complex and the concentration of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determined to be 9.7 microM in human neutrophils. Preformed filaments increase the rate of nucleation by WASp WA and the Arp2/3 complex but not the number of filaments that are generated. We propose that filament side binding by the Arp2/3 complex enhances its activation by WASp WA.
Higgs
H N
HN
Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Blanchoin
L
L
Pollard
T D
TD
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010441:Peptide Chain Elongation, Translational; D010446:Peptide Fragments / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D014923:Wiskott-Aldrich Syndrome / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein
C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D010446:Peptide Fragments; D011108:Polymers; D011506:Proteins; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein