IQGAP1 is a key node within the small GTPase network

Abstract

Coordination of the activity of multiple small GTPases is required for the regulation of many physiological processes, including cell migration. There are now several examples of functional interplay between small GTPase pairs, but the mechanisms that control GTPase activity in time and space are only partially understood. Here, we build on the hypothesis that small GTPases are part of a large, integrated network and propose that key proteins within this network integrate multiple signaling events and coordinate multiple small GTPase activities. Specifically, we identify the scaffolding protein IQGAP1 as a master regulator of multiple small GTPases, including Cdc42, Rac1, Rap1, and RhoA. In addition, we demonstrate that IQGAP1 promotes Arf6 activation downstream of β1 integrin engagement. Furthermore, following literature-curated searches and recent mass spectrometric analysis of IQGAP1-binding partners, we report that IQGAP1 recruits other small GTPases, including RhoC, Rac2, M-Ras, RhoQ, Rab10, and Rab5, small GTPase regulators, including Tiam1, RacGAP1, srGAP2 and HERC1, and small GTPase effectors, including PAK6, N-WASP, several sub-units of the Arp2/3 complex and the formin mDia1. Therefore, we propose that IQGAP1 acts as a small GTPase scaffolding platform within the small GTPase network, and recruits and/or regulates small GTPases, small GTPase regulators and effectors to orchestrate cell behavior. Finally, to identify other putative key regulators of small GTPase crosstalk, we have assembled a small GTPase network using protein-protein interaction databases.

Keywords: IQGAP1; Ras GTPases; signal transduction; small GTPase crosstalk; small GTPase network.

Figure 1. Gene Ontology analysis of IQGAP1-GFP pull-downs. Proteins enriched in the IQGAP1 pull-downs were mapped onto the GO category Biological Process (GOTERM_BP_FAT). Over-represented GO term (P < 0.05) were displayed as network-based enrichment maps (see Methods for details). Each node represents a GO term and each edge connects GO terms that contain at least one common protein. Node area is proportional to the number of proteins that belong to a particular GO term, and node color indicates the p value attributed to the enrichment of a particular GO term. Edge width is proportional to the similarity that exists between two GO terms. Nodes of this network were automatically organized using an algorithm that clusters nodes as a function of their connectivity and were then manually annotated.

Figure 2. IQGAP1 is a dual regulator of Arf6 and Rac1 downstream of integrin engagement. (A) The network of FN-induced adhesion complexes that connects β1 integrin to Rac1 and Arf6. Proteins identified in FN-induced adhesion complexes were mapped onto a literature-curated PPI network. Each node (circle) represents a protein (labeled with gene name) and each edge (line) represents a reported interaction between two proteins. Node color indicates whether a particular protein was also identified in and/or in. Node area is proportional to the normalized spectral count of proteins identified in. Reported direct binders of β1 integrin, Rac1 and Arf6 are displayed, and red edges highlight IQGAP1 as a selected putative link between β1 integrin, Rac1 and Arf6. To allow a clear visualization of the connection between β1 integrin, Arf6 and Rac1, nodes of this network were manually organized. (B and C) To study the role of IQGAP1 in Rac1 and Arf6 activation, mouse embryonic fibroblasts were treated with control oligonucleotide (siCTRL) or siRNA targeting IQGAP1 (siIQGAP1). Rac1 (B) and Arf6 (C) activity were measured using an effector pull-down approach. To compare activation profiles between experiments and during cell spreading on FN, Rac1 and Arf6 activity were normalized to that of siCTRL cells kept in suspension (B, n = 4; C, n = 6).

Figure 3. IQGAP1 recruits small GTPases, small GTPase regulators and small GTPase effectors to regulate cell behavior. Schematic representation of small GTPases, small GTPase regulators and small GTPase effectors known to co-purify with IQGAP1. Small GTPases are highlighted in red, small GTPase regulators in green and small GTPase effectors in blue.

Figure 4. IQGAP1 is a key node within the small GTPase network. To construct a small GTPase network, 144 out of the 162 known small GTPases were mapped onto literature-based protein-protein interaction (PPI) databases (18 small GTPases not found in the databases) and their known direct binding partners were selected. Each node represents a protein and each edge represents a reported interaction between 2 proteins. Nodes of this network were automatically organized using an algorithm that clusters nodes as a function of their connectivity. Small GTPases are highlighted in Red and IQGAP1 in yellow. Node size is proportional to the network topological parameter Stress. The stress of a node n is the number of shortest paths passing through n. A node has a high stress if it is traversed by a high number of shortest paths. Some nodes of interest were manually labeled.