Functional characterization of Wiskott-Aldrich syndrome protein and scar homolog (WASH), a bi-modular nucleation-promoting factor able to interact with biogenesis of lysosome-related organelle subunit 2 (BLOS2) and gamma-tubulin

J Biol Chem. 2010 May 28;285(22):16951-7. doi: 10.1074/jbc.M109.078501. Epub 2010 Mar 22.

Abstract

The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal gamma-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.

MeSH terms

  • Actin-Related Protein 2-3 Complex / metabolism*
  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Gene Expression Regulation*
  • Lysosomes / metabolism*
  • Mice
  • Microtubule-Associated Proteins / metabolism*
  • Models, Biological
  • Nerve Tissue Proteins / metabolism*
  • PC12 Cells
  • Proteins / metabolism*
  • Rats
  • Tubulin / metabolism*
  • Wiskott-Aldrich Syndrome Protein / metabolism*

Substances

  • Actin-Related Protein 2-3 Complex
  • BLOC1S2 protein, human
  • BLOC1S2 protein, rat
  • Microtubule-Associated Proteins
  • Nerve Tissue Proteins
  • Proteins
  • Tubulin
  • Wiskott-Aldrich Syndrome Protein