The verprolin-like central (vc) region of Wiskott-Aldrich syndrome protein induces Arp2/3 complex-dependent actin nucleation.
Wiskott-Aldrich Syndrome protein (WASp) and related proteins stimulate actin filament nucleation by Arp2/3 complex. The isolated C-terminal VCA domain of WASp (containing Verprolin-like, Central and Acidic regions) is constitutively active but autoinhibited in the full-length protein. This study compared the ability of parts of VCA fused to the C terminus of glutathione S-transferase (GST) to bind actin and Arp2/3 complex in vitro and to activate actin polymerization in vitro and in cells. Fluorescence anisotropy measurements showed that GST-CA and GST-A bound Arp2/3 complex with K(d) values of 0.11 microm and 1.0 microm, respectively, whereas GST-VC displayed almost undetectable binding (K(d) > 1 mm). However, GST-VC activated actin nucleation through Arp2/3 complex in vitro, though requiring 70-fold higher concentration than GST-VCA while neither GST-CA nor GST-A activated Arp2/3 complex in vitro, though both GST-CA and GST-A inhibited Arp2/3 complex activation by WASp VCA. None of these constructs bound WASp from macrophage lysates. Both GST-VC and GST-CA induced actin accumulations when microinjected into primary human macrophages or human endothelial vein cells. However, only microinjection of GST-VC led to a significant increase of cellular polymerized actin. Additionally, endogenous Arp2/3 complex, but not WASp, colocalized with these GST-VC-induced actin accumulations. These data suggest that WASp constructs lacking the A region, previously thought to be indispensable for actin nucleation, are able to bind and activate Arp2/3 complex in vitro and in vivo.
Hüfner
K
K
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten and Max von Pettenkofer-Institut für Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, Pettenkoferstr. 9, 80336 München, Germany.
Higgs
H N
HN
Pollard
T D
TD
Jacobi
C
C
Aepfelbacher
M
M
Linder
S
S
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000502:physiology*; D008845:Microinjections; D011506:Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; 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; D005656:Fungal Proteins; D008840:Microfilament Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C090350:VRP1 protein, S cerevisiae; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D005982:Glutathione Transferase