Activation by Cdc42 and Pip2 of Wiskott-Aldrich Syndrome Protein (Wasp) Stimulates Actin Nucleation by Arp2/3 Complex

doi:10.1083/jcb.150.6.1311

Published online 18 September 2000. doi:10.1083/jcb.150.6.1311

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© The Rockefeller University Press, 0021-9525/2000//1311 $5.00
The Journal of Cell Biology, Volume 150, Number 6, , 2000 1311-1320

Original Article

Activation by Cdc42 and Pip₂ of Wiskott-Aldrich Syndrome Protein (Wasp) Stimulates Actin Nucleation by Arp2/3 Complex

Henry N. Higgs^a and Thomas D. Pollard^a

^a The Salk Institute for Biological Studies, La Jolla, California 92037
The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037.(858) 452-3683(858) 453-4100 x1716

We purified native WASp (Wiskott-Aldrich Syndrome protein) frombovine thymus and studied its ability to stimulate actin nucleationby Arp2/3 complex. WASp alone is inactive in the presence orabsence of 0.5 µM GTP-Cdc42. Phosphatidylinositol 4,5bisphosphate (PIP₂) micelles allowed WASp to activate actinnucleation by Arp2/3 complex, and this was further enhancedtwofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complexand WASp in the presence of PIP₂ and Cdc42 concentrated aroundlipid micelles and vesicles, providing that Cdc42 was GTP-boundand prenylated. Thus, the high concentration of WASp in neutrophils(9 µM) is dependent on interactions with both acidic lipidsand GTP-Cdc42 to activate actin nucleation by Arp2/3 complex.The results also suggest that membrane binding increases thelocal concentrations of Cdc42 and WASp, favoring their interaction.

Key Words: prenylation • membrane • GBD • neutrophil • thymus

Abbreviations used in this paper: CF-PE, carboxyfluorescein-labeledPE; GBD, GTPase binding domain; GST, glutathione-S-transferase;HSS, high speed supernatant; PA, phosphatidic acid; PC, phosphatidylcholine;PE, phosphatidylethanolamine; PIP₂, phosphatidylinositol 4,5bisphosphate; PS, phosphatidylserine; PMN, polymorphonuclearleukocytes; WA, WASp/Scar protein COOH-terminal 70–100amino acids; WASp, Wiskott-Aldrich Syndrome protein.

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