Purification of bacterially expressed human IQGAP1 and characterization of its interactions with various ligands. a, recombinant His₆-tagged IQGAP1 was produced in E. coli and purified by affinity chromatography on Ni-Sepharose and CaM-Sepharose. IQGAP1 was eluted from CaM Sepharose with 10 mm EGTA and 0.5 m KSCN. The yield was typically 0.4 mg per liter of culture. Samples at the different steps of the purification were analyzed on SDS-PAGE and Coomassie Blue staining. b, IQGAP1 binds Rac1 and Cdc42 in a CaM-sensitive fashion. His₆-tagged IQGAP1 (180 nm) coprecipitation assays with GST (1 μm), GST-Rac1-GTPgS (1 μm), or GST-Cdc42-GTPgS (1 μm) in the absence or presence of CaM (5 μm). Bound fractions were analyzed by Western blot using an anti-IQGAP1 antibody. c, IQGAP1 binds BR-CRIB (wt) and to a lesser extent BR-CRIB (H211D) of N-WASP in a CaM-sensitive fashion. His-tagged IQGAP1 (120 nm) coprecipitation assays with GST (1 μm) or GST-BR-CRIB wt (1 μm) or GST-BR-CRIB (H211D) in the absence or presence of CaM (5 μm). Bound fractions were analyzed by Western blot as in panel b. d, map of full-length N-WASP and the BR-CRIB fragment with the position of the mutation H211D.

IQGAP1 is a barbed end capper. a, IQGAP1 blocks barbed end growth. Barbed end growth of 2 μm MgATP-G-actin (10% pyrenyl-labeled) was measured in the presence of 105 pm spectrin-actin seeds (SP) and the indicated concentrations of IQGAP1. Inset, IQGAP1 does not affect pointed end growth. 2.5 μm MgATP-G-actin (10% pyrenyl-labeled) was polymerized with 10 nm gelsolin-actin (GA₂) complexes in the presence or absence of 300 nm IQGAP1. b, initial rates of barbed end growth corresponding to data in a (+ additional data points) were plotted versus the concentration of IQGAP1. The best fit of the data gave a K_B of 34 nm. c, IQGAP1 caps ADP-bound barbed ends with a higher affinity than ADP-Pi-bound barbed ends. Dilution-induced depolymerization of 60 nm F-actin (10% pyrenyl-labeled) was measured in the presence of the indicated concentrations of IQGAP1. d, initial rates of depolymerization corresponding to the data in c were plotted versus the concentration of IQGAP1. The best fit curve was calculated with a K_d of 4 nm. e and f, CaM inhibits barbed end capping by IQGAP1. Barbed end growth was measured in the presence of 105 pm spectrin-actin seeds (SP), 2 μm MgATP-G-actin (10% pyrenyl-labeled), and the indicated concentrations of IQGAP1 and Ca-CaM (red) or apo-CaM (blue). The best fit of the data gave the same K_d value of 30 nm for binding of apo- and holo-CaM to IQGAP1. g, initial rates of barbed end growth corresponding to the data in e, f were plotted versus the concentration of Ca-CaM (red) and apo-CaM (blue). The curve is calculated with a K_C of 40 nm for the IQGAP1-CaM complex.

Localization of the barbed end capping activity of IQGAP1. a, N-terminal fragment (residues 1–216) that does not contain the dimerizing coiled coil domain does not inhibit barbed end growth. The N-terminal fragment comprising the CH domain (1–522) inhibits barbed end polymerization with low affinity. A C-terminal fragment of IQGAP1 (744–1657) inhibits barbed end polymerization with an affinity close to the full-length protein. 2.5 μm MgATP-G-actin (10% pyrenyl-labeled) was polymerized in the presence of 110 pm SP seeds and the indicated concentrations of the various IQGAP1 fragments. b and c, IQGAP1 fragment 744–1657 inhibits actin assembly at barbed ends with a value of K_B of 68 nm. 2.5 μm MgATP-G-actin (10% pyrenyl-labeled) was polymerized in the presence of 105 pm SP seeds and the indicated concentrations of the IQGAP1 fragment 744–1657. d and e, CaM relieves barbed end capping by the IQGAP1 fragment 744–1657. Barbed end growth was measured in the presence of 110 pm spectrin-actin seeds (SP), 2 μm MgATP-G-actin (10% pyrenyl-labeled), and the indicated concentrations of IQGAP1-(744–1657) and Holo-CaM. For clarity, only some data points are shown. f, SDS-PAGE of the various recombinant IQGAP1 fragments purified and used in this work. g, diagram showing the structural organization of IQGAP1, its identified binding partners, and the fragments used in a and b.

IQGAP1 does not activate N-WASP. a, IQGAP1 does not affect the catalysis of filament branching reaction by constitutively active VCA. 2.5 μm MgATP-G-actin (10% pyrenyl-labeled) was polymerized in the presence of 110 nm VCA, 38 nm Arp2/3 complex and the indicated concentrations of IQGAP1. b, IQGAP1 does not activate N-WASP. Higher extent of inhibition because of barbed end capping is recorded at higher concentrations of IQGAP1. 2.5 μm MgATP-G-actin (10% pyrenyl-labeled) was polymerized in the presence of 40 nm N-WASP and 38 nm Arp2/3 complex and the indicated concentrations of IQGAP1. Inset, maximum polymerization speed is plotted against the concentration of IQGAP1. c, elution profile of the size exclusion chromatography of IQGAP1 fragment 962–1345. d, spectra of eluted peak fractions from panel c, and of supernatant of fractions in peak 1 after boiling for 5 min and centrifugation. e, SDS-PAGE analysis of IQGAP1 fragment 962–1345 purified by size exclusion chromatography. f, functional analysis of the fractions of peak 1 and peak 2 for the ability to activate N-WASP, before and after boiling. Fractions of peak 1 activate N-Wasp both before and after boiling. Fractions of peak 2 do not activate N-WASP. 2.5 μm MgATP-G-actin (10% pyrenyl-labeled) was polymerized in the presence of 24 nm N-WASP, 38 nm Arp2/3 complex, and protein at the same concentration of 110 nm (according to UV absorbance spectra) or equal volume of supernatant of boiled sample as indicated. g, nucleic acids activate N-WASP to stimulate polymerization into branched filaments. 2.5 μm MgATP-G-actin (10% pyrenyl-labeled) was polymerized in the presence of 24 nm N-WASP, 38 nm Arp2/3 complex, and the indicated concentrations of various nucleic acids. Controls showed that the same amounts of nucleic acids do not affect polymerization of actin alone. The standard activation by PIP2 is shown.

Scheme for regulation of several activities of IQGAP1 by Ca-CaM. Holo-CaM inhibits IQGAP1 interaction with its other partners by binding to its IQ-motifs. In the absence of CaM, IQGAP1 adopts an “open” conformation for binding by Cdc42, Rac1, and other partners. Cdc42 and BR-CRIB of N-WASP bind IQGAP1 synergistically.