posted on 2007-03-20, 00:00authored byNenad Tomasevic, Zhiheng Jia, Alan Russell, Toby Fujii, James J. Hartman, Sheila Clancy, Manping Wang, Christophe Beraud, Kenneth W. Wood, Roman Sakowicz
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.