Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex

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 microM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP(2)) 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 PIP(2) 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 microM) 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.

Figure 1

Extraction and purification of WASp from leukocytes. (A) Schematic diagram of WASp with the domains and constructs used in this study. (B) Extraction of WASp. Homogenates (Homog.) from human peripheral neutrophils and bovine thymus were fractionated into a 4,000-g pellet (LSP), 100,000-g pellet (HSP), and 100,000-g supernatant (HSS). 10 μg of the total homogenate and equal volumes of the centrifuged fractions were Western blotted with AB1. (C) Determination of WASp concentration in human neutrophils. SDS-PAGE of homogenates at various dilutions in the absence or presence of varying concentrations of WASp152-309. Western blots against AB1 were analyzed by densitometry. (D) Gel filtration of purified WASp on Superdex200 in 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, and 1 mM DTT. The concentrations of WASp in the eluted fractions were measured by quantitative Western blotting. (inset) Coomassie blue–stained SDS-PAGE of 0.2 μg purified WASp before gel filtration.

Figure 2

Activation of Arp2/3 complex by native WASp and WASp-WA. (A) Effect of Cdc42 and PIP₂ on WASp activation of Arp2/3 complex. Monomeric MgATP actin (4 μM, 5% pyrene-labeled) was polymerized in the presence of 10 nM of Arp2/3 complex and no addition (open circles), 4 nM WASp (filled circles), 4 nM WASp, and 500 nM prenylated GTPγS-Cdc42 (open diamonds), 4 nM WASp, and 20 μM PIP₂ micelles (open squares), 4 nM WASp, 20 μM PIP₂ micelles and 500 nM prenylated GTPγS-Cdc42 (filled triangles), and 500 nM WASp WA (crosses). (B) Dependence of nucleation by Arp2/3 complex and native WASp on PIP₂ concentration. Conditions were as described in A, with 4 nM WASp and a varying concentration of PIP₂ in the absence of Cdc42. The concentration of filament ends was calculated from the slopes of polymerization curves at 80% polymerization. (C) Dependence of nucleation by Arp2/3 complex on the concentrations of dimeric GST-WA (closed circles) or monomeric WASp-WA (open circles). Conditions are as in A, except no PIP₂ or Cdc42 was added.

Figure 3

Fluorescence micrographs of the products of actin polymerization reactions. Polymerization reactions contained 4 μM monomeric MgATP actin, with or without 100 nM of Arp2/3 complex, 4 nM WASp, 0.5 μM prenylated GTPγS-Cdc42, 22 μM PIP₂ micelles, or 100 μM of phospholipid vesicles. After polymerization for 20 min at 23°C in KMEI containing 4 μM rhodamine-phalloidin, samples were diluted 625-fold (A–F) or 25-fold (G and H) into motility buffer, mounted on nitrocellulose-coated coverslips, and viewed with filters for rhodamine. (A) Arp2/3 complex, prenylated GTPγS-Cdc42, PIP₂ micelles, (B) WASp, prenylated GTPγS-Cdc42, PIP₂ micelles, (C) Arp2/3 complex, WASp, (D) Arp2/3 complex, WASp, PIP₂ micelles, (E) Arp2/3 complex, WASp, prenylated GTPγS-Cdc42, (F) Arp2/3 complex, WASp, prenylated GTPγS-Cdc42, PIP₂ micelles, (G) Arp2/3 complex, WASp, prenylated GTPγS-Cdc42, multilamellar vesicles containing 50% cholesterol/14.5% PC/14.5% PE/20% PS/1% CF-PE, or (H) Arp2/3 complex, WASp, prenylated GTPγS-Cdc42, multilamellar vesicles containing 50% cholesterol/19.5% PC/19.5% PE/10% PIP₂/1% CF-PE. Inset in G shows corresponding fluorescein image of G, showing the vesicle at the interior of the actin halo (left) and a vesicle not surrounded by actin.

Figure 5

WASp152-309 interacts with GTPγS-Cdc42 but not with PIP₂. (A) Effect of GTPγS-Cdc42 and PIP₂ on nucleation inhibition by WASp152-309. Nucleation assays were performed as described in Fig. 2 A with 10 nM of Arp2/3 complex alone (filled circles); Arp2/3 complex, 250 nM WASp WA (open circles); Arp2/3 complex, WASp WA, 3 μM WASp152-309 (filled squares); Arp2/3 complex, WASp WA, WASp152-309, 1.6 μM prenylated GTPγS-Cdc42 (open squares); Arp2/3 complex, WASp WA, WASp152-309, 3.2 μM prenylated GTPγS-Cdc42 (filled diamonds); and Arp2/3 complex, WASp WA, WASp152-309, 30 μM PIP₂ (open diamonds). (B) Effect of WASp152-309 on full-length native WASp activation by PIP₂ and GTPγS-Cdc42. Nucleation assays were performed as described in Fig. 2 A with 10 nM of Arp2/3 complex alone (open circles); Arp2/3 complex, 2 nM WASp (filled circles); Arp2/3 complex, WASp, 20 μM PIP₂ (open squares); Arp2/3 complex, WASp, PIP₂, 25 μM WASp152-309 (filled squares); Arp2/3 complex, WASp + PIP₂, 0.5 μM prenylated GTPγS-Cdc42 (filled triangles); and Arp2/3 complex, WASp, PIP₂, prenylated GTPγS-Cdc42, 25 μM WASp152-309 (open triangles).

Figure 4

Regulation of WASp-WA by a fragment of WASp including the GBD. (A) WASp 152-309 inhibits Arp2/3 complex activation by WASp WA. Nucleation reactions included 4 μM of monomeric MgATP actin (5% pyrene-labeled), 10 nM of Arp2/3 complex, 250 nM WASp WA or Scar1 WA, and varying concentrations of WASp 152-309. The concentration of filament ends created by Arp2/3 complex was calculated from the slope of the polymerization curve at 80% polymerization. Percent nucleation is the concentration of ends created relative to that created by Arp2/3 complex and WASp WA or Scar1 WA in the absence of WASp 152-309, after subtraction of the concentration of filaments created in the absence of WA. (B) Binding of WASp 152-309 to WASp WA was measured by fluorescence anisotropy of rhodamine-labeled WASp WA (0.1 μM). The calculated dissociation equilibrium constant is 0.42 μM. (C) Fluorescence anisotropy assay for competition between Arp2/3 complex and WASp152-309 for binding WASp WA. Rhodamine-labeled WASp WA (0.1 μM) has an anisotropy of 0.162 when bound to 1 μM of Arp2/3 complex. The smaller WASp152-309 competes off Arp2/3 complex, giving an anisotropy of 0.132.