IQGAP1 Regulates Cell Motility by Linking Growth Factor Signaling to Actin Filament Assembly
Benseñor, Lorena Betsabé, Department of Biology, University of Virginia
Bloom, George, Department of Biology, University of Virginia
IQGAP1 is a cytosolic protein that integrates intracellular signals that regulate the actin cytoskeleton reorganization and cell motility. IQGAP1 binding partners in this complex regulatory pathway includes filamentous actin, Cdc42 and Rac1, E-cadherin and ß-catenin and microtubule binding proteins APC and CLIP-170. Two aspects of IQGAP1 function were addressed in this work. First, the minimal region of IQGAP1 regulating actin assembly and cell migration downstream of growth factor receptor activation was studied in mammalian cultured cells and in vitro assays. One intriguing aspect of IQGAP1 is that it can bind F-actin in vitro, whereas in intact cells it co-localizes with actin filaments in the cortex, but not in stress fibers. This observation suggests that IQGAP1 is actively recruited to the subcortical region of the plasma membrane, but the domains and/or binding partners required for IQGAP1 targeting are unknown. To elucidate whether IQGAP1 contains a putative targeting domain I generated N-terminus mammalian expression IQGAP1 deletion mutants. I analyzed these fragments for their ability to co-localize with F-actin at the leading edge of motile cells and their effect on lamellipodial dynamics when overexpressed in cultured cells. Here we show that an N-terminal fragment of IQGAP1 containing the CHD is required and sufficient for targeting IQGAP1 to the cell cortex. In addition, lamellipodia protrusions are impaired in cells overexpressing the small mutant protein of IQGAP1. Overexpression or downregulation of IQGAP1 increases or decreases cell migration, respectively, but the underlying molecular mechanisms are not well understood. Here we present evidence that IQGAP1 stimulates branched actin filament assembly, which provides the force for lamellipodial protrusion. Binding of FGF2 to FGFR1 regulates this function of IQGAP1. Stimulation with FGF2 triggered: IQGAP1-dependent lamellipodial protrusions and cell migration; intracellular associations of IQGAP1 with FGFR1, Arp2/3 iii complex and N-WASP and recruitment of IQGAP1, FGFR1, N-WASP and Arp2/3 complex to lamellipodia. In vitro, IQGAP1 bound directly to the cytoplasmic tail of FGFR1 and to N-WASP, and stimulated branched actin filament nucleation in the presence of N- WASP and Arp2/3 complex. These results conclude that IQGAP1 links FGF2 signaling to Arp2/3 complex-dependent actin assembly by binding FGFR1 and activating N- WASP.
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PHD (Doctor of Philosophy)
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