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Figure 1. Sequence determinants of the interaction between the NH2- and COOH-terminal regions of N-WASP. (A) A summary of the nomenclature and amino acid boundaries of the various domains and fragments of bovine N-WASP described in this study. (B) GST-VCA (or GST alone as a control) immobilized on glutathione-Sepharose beads was tested for its ability to pull down various fragments of N-WASP (shown in A) produced as Myc-tagged (MT), 35S-labeled proteins. (C) Dose-dependent binding of the indicated N-WASP fragments to GST-VCA beads. The slope of the line defined by each set of points is inversely proportional to the dissociation constant. (D) GST fusions of the indicated subfragments of the VCA segment of N-WASP (defined in A) were immobilized on glutathione-Sepharose beads and used to pull down the MT-WG fragment from the NH2 terminus of N-WASP. In B and D, 5% of the input and 33% of the pulled down material was analyzed on a 5â15% SDSâpolyacrylamide gel.
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Figure 2. The NH2-terminal domain of N-WASP inhibits binding of Arp2/3 complex to the COOH-terminal VCA segment in a Cdc42- and PI(4,5)P2-regulated manner. (A) Purified protein preparations of the hexahistidine-tagged (HT) WG and WGP fragments and GST-VCA used in this study are shown on a 5â15% SDSâpolyacrylamide gel. (B) Binding of GST-VCA or GST-fused full-length N-WASP (0.5 μM each) to Arp2/3 complex (0.1 μM) was assayed by incubating the components in solution and capturing the GST fusion protein on glutathione-Sepharose beads. The amount of Arp2/3 bound was determined by analyzing 50% of the input and 37.5% of the pulled down material on an α-Arp2 immunoblot. (C) Binding of GST-VCA (0.2 μM) to Arp2/3 (0.1 μM) was tested (as in B) in the presence of the indicated concentrations of HT-WG or HT-WGP. Immunoblotting was used to assay the amount of GST-VCA (α-GST), Arp2/3 (α-Arp3 and α-p34Arc), and HT-WG or HT-WGP (α-pentaHis) precipitated on the glutathione-Sepharose beads. (D) The ability of HT-WG (2 μM) to inhibit the binding of Arp2/3 (0.1 μM) to GST-VCA (0.2 μM) was tested in the presence of GTPγS-loaded Cdc42 (5 μM) or PI(4,5)P2-containing vesicles (100 μM; PI(4,5)P2/PI/PC = 10:45:45). (E) GST-Cdc42 beads, loaded with either GTPγS or GDP, were tested for their abilities to bind to Arp2/3 (1 μM) in the presence of N-WASP (1 μM) and PI(4,5)P2-containing vesicles (100 μM). Arp2/3 pulled down by GST-Cdc42 beads in the absence of any N-WASP represents nonspecific, background binding under the conditions of the assay. For the α-Arp2 immunoblot, 7.5% of the input, 10% of the material pulled down on the GST-Cdc42 beads, and (as a control for comparison) 10% of the material pulled down by GST-VCA (1 μM) on glutathione- Sepharose beads was loaded on the gel.
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Figure 3. The NH2-terminal domain of N-WASP does not inhibit binding of G-actin to the VCA segment. GST pull-down assays were used to measure the binding of actin (0.05 μM) to GST-CA, GST-N-WASP, GST-VCA (0.5 μM each), or to GST-VCA (0.5 μM) in the presence of HT-WG or HT-WGP (2.5 μM each). The amount of actin bound was compared using an α-actin immunoblot, loading 100% of the input and 40% of the pulled down material. The amounts of GST-N-WASP, GST-CA, GST-VCA, HT-WG, and HT-WGP pulled down were compared by immunoblotting with α-GST or α-pentaHis.
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Figure 4. The NH2-terminal domain of N-WASP inhibits Arp2/3-mediated actin nucleation activity of the VCA segment in a Cdc42, and PI(4,5)P2-regulated manner. (A) The pyrene actin assay was used to monitor the polymerization of 1.3 μM G-actin (1 μM unlabeled actin + 0.3 μM pyrene labeled actin) in the presence of Arp2/3 (30 nM), GST-VCA (10 nM), and the indicated concentrations of HT-WG. (B) The ability of GST-VCA (10 nM or 50 nM) to activate Arp2/3 complex (30 nM) was measured in the presence of increasing concentrations of HT-WG. The maximum polymerization rate, calculated from the linear phase of polymerization curves of the type shown in A, is taken as a measure of Arp2/3 activation. (C) Cdc42-GTPγS can stimulate actin polymerization (1.3 μM actin, 30 nM Arp2/3) in the presence of GST-VCA (5 nM) and HT-WG (200 nM) more effectively than Cdc42-GDP. (D) The ability of either Cdc42-GTPγS (200 nM), PI(4,5)P2-containing vesicles (10 μM), or both together to stimulate actin polymerization (1.3 μM actin, 30 nM Arp2/3 complex) in the presence of GST-VCA (5 nM) and HT-WG (200 nM).
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Figure 5. Hydrodynamic characterization of N-WASP. (A) A purified preparation of untagged rat N-WASP used for analytical ultracentrifugation and gel filtration chromatography is shown on a 10% SDSâpolyacrylamide gel. (B) A radial absorbance plot showing the equilibrium distribution of N-WASP (0.55 mg/ml, 10 μM) after sedimentation in an analytical ultracentrifuge. The solid line indicates the best fit to the data, assuming a single component model, and the top panel shows residuals of the fit. (C) Elution profile from gel filtration chromatography of N-WASP on an analytical grade Superdex 200 column. The volumes at which various standard proteins elute are indicated.
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Figure 6. The WH1 domain is not required for the ability of PI(4,5)P2 to activate N-WASP. (A) A 10% SDSâpolyacrylamide gel showing purified protein preparations of GST-tagged wild-type N-WASP (GST-N-WASP) or a GST-tagged truncation mutant of N-WASP lacking the WH1 domain (GST-(ÎWH1) N-WASP). (B) Actin polymerization (1.3 μM actin, 30 nM Arp2/3 complex) in the presence of the indicated combinations of GST-N-WASP (50 nM), GST-(ÎWH1)N-WASP (50 nM), Cdc42-GTPγS (100 nM), or PI(4,5)P2-containing vesicles (1 μM).
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Figure 7. The BR in the NH2 terminus of N-WASP specifically binds to PI(4,5)P2. (A) Diagram showing various mutants of the GBR of bovine N-WASP, which includes the BR and the Cdc42/Rac interactive binding (CRIB) motif. In GBR4, the four Xs denote the substitution of four lysine residues (numbers 186, 189, 192, and 195) with glutamic acid residues. (B) Binding of the GST-GBR mutants shown in A to [3H]phosphatidylcholine-labeled vesicles of the indicated compositions. A GST fusion of the β spectrin pleckstrin homology domain (GST-SpecPH) was used as a positive control in the assay.
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Figure 8. The BR in the NH2 terminus is required for the ability of PI(4,5)P2 to activate N-WASP. (A) A 10% SDSâpolyacrylamide gel showing purified protein preparations of wild-type GST-N-WASP or a GST-tagged mutant of N-WASP containing an internal deletion of the BR (GST-(ÎB)N-WASP). (B) The maximum rate of polymerization was used to quantitate Arp2/3 activity (1.3 μM actin, 30 nM Arp2/3 complex) in the presence of two different concentrations of GST-N-WASP or GST-(ÎB) N-WASP and the indicated combinations of Cdc42-GTPγS (450 nM) and PI(4,5)P2-containing vesicles. While the data shown in B is at 1 μM lipid, GST-(ÎB)N-WASP was PI(4,5)P2-insensitive at higher lipid concentrations as well (up to 5 μM).
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Figure 9. N-WASP is required for PI(4,5)P2-stimulated actin polymerization in Xenopus HSS. (A) Affinity-purified α-N-WASP antibody or antibody buffer (mock) was used to immunodeplete N-WASP from Xenopus HSS, and the material in the supernatant was analyzed for the presence of N-WASP by immunoblotting using the same α-N-WASP antibody. (B) Comparison of actin assembly stimulated by 100 μM PI(4,5)P2-containing vesicles in mock-depleted HSS, α-N-WASPâdepleted HSS, and in depleted HSS reconstituted with 50 nM recombinant N-WASP. The add back of recombinant N-WASP to α-N-WASPâdepleted extracts and the addition of PI(4,5)P2-containing vesicles was performed at the time points indicated by the arrows. (C and D) The abilities of GST-N-WASP, GST-(ÎWH1)N-WASP, or GST-(ÎB)N-WASP (100 nM each) to restore PI(4,5)P2-stimulated actin polymerization to HSS depleted of endogenous N-WASP.
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Figure 10. Two models for N-WASP activation. A black circle has been placed between sequence elements or proteins that have been shown to physically interact.
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