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J. Cell Biol. 150 (6): 1311-1320

Copyright © 2000 by the Rockefeller University Press.

Original Article

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

Henry N. Higgsa, and Thomas D. Pollarda

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

Abstract: We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 µM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP2) micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP2 and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42 was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 µM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction.

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

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

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Lateral Sequestration of Phosphatidylinositol 4,5-Bisphosphate by the Basic Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Is Due to Nonspecific Electrostatic Interactions.
J. Wang, A. Gambhir, G. Hangyas-Mihalyne, D. Murray, U. Golebiewska, and S. McLaughlin (2002)
J. Biol. Chem. 277, 34401-34412
   Abstract »    Full Text »    PDF »
Remodeling of organelle-bound actin is required for yeast vacuole fusion.
G. Eitzen, L. Wang, N. Thorngren, and W. Wickner (2002)
J. Cell Biol. 158, 669-679
   Abstract »    Full Text »    PDF »
Normal Arp2/3 complex activation in platelets lacking WASp.
H. Falet, K. M. Hoffmeister, R. Neujahr, and J. H. Hartwig (2002)
Blood 100, 2113-2122
   Abstract »    Full Text »    PDF »
CHEMOTACTIC SIGNALING PATHWAYS IN NEUTROPHILS: FROM RECEPTOR TO ACTIN ASSEMBLY.
G. Cicchetti, P. G. Allen, and M. Glogauer (2002)
Critical Reviews in Oral Biology & Medicine 13, 220-228
   Abstract »    Full Text »    PDF »
Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.
A. V. HUBBERSTEY and E. P. MOTTILLO (2002)
FASEB J 16, 487-499
   Abstract »    Full Text »    PDF »
The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices.
K. J. Bayless and G. E. Davis (2002)
J. Cell Sci. 115, 1123-1136
   Abstract »    Full Text »    PDF »

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