Title: IQGAP1 Scaffolding Connects EGFR and Phosphoinositide Signaling to Cytoskeletal Reorganization
Abstract: IQGAP1 is a multi-domain protein that acts as a scaffold for multiple signaling pathways, especially in cancer cells where it is seen to be overexpressed. While IQGAP1 is known to generate the lipid messenger PI(3,4,5)P3 by scaffolding the phosphoinositide kinases PIPKIs and PI3K, the dynamics of this scaffold in intact, living cells remains unknown. Here, we delineate the spatial and temporal effects of IQGAP1 signaling in live cells under basal and stimulated conditions using biophysical techniques. Specifically we use fluorescence lifetime imaging to study protein-protein interactions through FRET, and Number and Brightness technique which studies molecular number and aggregation in an observation volume. We observe EGF-mediated changes in both IQGAP1 and EGFR distribution and see that IQGAP1 is involved in EGFR clustering. We also demonstrate that IQGAP1 interacts strongly with PIPKIγ at intracellular compartments and at the plasma membrane, and that it scaffolds PI3K and PIPKIγ in response to certain physiological changes. Additionally, we show that IQGAP1 scaffolds phosphoinositides with PI3K, PIPKIγ and EGFR, and forms clusters upon cell stimulation with EGF. Importantly, we show that IQGAP1 connects PI(3,4,5)P3-mediated signaling and cytoskeletal signaling pathways by binding PIPKIγ in proximity of the cytoskeletal proteins talin and Cdc42. Our results support a model in which IQGAP1 mediates crosstalk between EGFR receptor activation, phosphoinositide signaling and the cytoskeleton.