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Fig. 8. IQGAP1 fragments stimulate actin assembly and bind N-WASP. (A) A schematic representation of functional domains present in recombinant full-length IQGAP1 and four fragments used for experiments documented here: CHD, F-actin-binding calponin homology domain; IR, IQGAP repeats that mediate homodimerization; WW, ERK2-binding WW domain; IQ, calmodulin-binding IQ motifs; GRD, GAP (GTPase activating protein)-related domain involved in binding activated Cdc42 and Rac1. All proteins were his-tagged at their N termini, and the diagrams indicate whether each protein is a monomer or homodimer in aqueous solution. (B) The pyrene actin assembly assay was used to evaluate each protein at several concentrations in the presence of 1.3 µM actin (5% pyrene-labeled), 50 nM N-WASP and 50 nM Arp2/3 complex. Shown here are the maximum velocities (V_max) of actin assembly (upper panels) and lag times before V_max was reached (lower panels). Note that optimal concentrations of all recombinant proteins, except IQGAP1_NT, supported a V_max approx. twofold higher than controls that contained only actin, N-WASP and Arp2/3 complex, but that the optimal concentration of full-length IQGAP1 (IQGAP1_FL) reached V_max at least twice as fast as the fragments. (C) N-WASP was mixed with nickel-agarose beads or nickel-agarose beads that were pre-loaded with recombinant, his-tagged IQGAP1_FL, IQGAP1_NT, IQGAP1_2-522, IQGAP1_2-210, IQGAP1_2-71, or tau as a negative control. Beads contained an approx. twofold molar excess of his-tagged proteins relative to N-WASP, and chemiluminescent immunoblotting was used to detect any N-WASP that may have bound to beads. Note the specific binding of N-WASP to all forms of IQGAP1 that were tested. The slightly increased electrophoretic mobility of N-WASP in the IQGAP1_2-522 pull-down assay probably represents a gel artefact caused by that fact N-WASP and IQGAP1_2-522 migrate nearly identically in SDS-PAGE.

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