During the fiscal year we accomplished the following: 1. During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. We showed that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knockout of endogenous IQGAP1 significantly increased the formation of nuclear YAP/TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP/TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling. 2. Helicobacter pylori infection is responsible for gastric carcinogenesis, but host factors also contribute. We showed that 1 year after infection with H. pylori, IQGAP1+/- mice developed more preneoplastic lesions and 8-fold more gastrointestinal neoplasia than littermate control mice. In vitro, IQGAP1 knockdown favored the acquisition of a mesenchymal phenotype and cancer stem cell-like properties induced by H. pylori infection. These findings indicate that alterations in IQGAP1 signaling promote the emergence of cancer stem cells and the development of gastric adenocarcinoma in the context of H. pylori infection. 3. In signaling, Rho-family GTPases bind effector proteins and alter their behavior. We solved the crystal structure of Cdc42GTP bound to the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP2. Four molecules of Cdc42 are bound to two GRD molecules, which bind each other in a parallel dimer. Two Cdc42s bind very similarly to the Ras/RasGAP interaction, while the other two bind primarily to extra domain sequences from both GRDs, tying the GRDs together. Calorimetry confirmed two-site binding of Cdc42GTP for the GRDs of both IQGAP2 and IQGAP1. Mutation of important extra domain residues reduced binding to single-site and abrogated Cdc42 binding to a much larger IQGAP1 fragment. Importantly, Rac1GTP displays only single-site binding to the GRDs, indicating that just Cdc42 promotes IQGAP dimerization. The structure identifies an unexpected role for Cdc42 in protein dimerization thus expanding the repertoire of interactions of Ras family proteins with their targets. 4. The intracellular bacterial pathogen Shigella infects and spreads through the human intestinal epithelium. Effector proteins delivered by Shigella into cells promote infection by modulating diverse host functions. We demonstrated that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of S. flexneri spread through cell monolayers. We showed that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrated that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.

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Ozdemir, E Sila; Jang, Hyunbum; Gursoy, Attila et al. (2018) Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2. J Biol Chem 293:3685-3699
Zhang, Mingzhen; Li, Zhigang; Wang, Guanqiao et al. (2018) Calmodulin (CaM) Activates PI3K? by Targeting the ""Soft"" CaM-Binding Motifs in Both the nSH2 and cSH2 Domains of p85?. J Phys Chem B :
Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y et al. (2018) Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry. J Am Soc Mass Spectrom 29:1721-1737
Li, Zhigang; Zhang, Yonghong; Hedman, Andrew C et al. (2017) Calmodulin Lobes Facilitate Dimerization and Activation of Estrogen Receptor-?. J Biol Chem 292:4614-4622
Chawla, Bhavna; Hedman, Andrew C; Sayedyahossein, Samar et al. (2017) Absence of IQGAP1 Protein Leads to Insulin Resistance. J Biol Chem 292:3273-3289
LeCour Jr, Louis; Boyapati, Vamsi K; Liu, Jing et al. (2016) The Structural Basis for Cdc42-Induced Dimerization of IQGAPs. Structure 24:1499-508
Sayedyahossein, Samar; Li, Zhigang; Hedman, Andrew C et al. (2016) IQGAP1 Binds To YAP and Modulates Its Transcriptional Activity. J Biol Chem :
Choi, Suyong; Hedman, Andrew C; Sayedyahossein, Samar et al. (2016) Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases. Nat Cell Biol 18:1324-1335
Bessède, Emilie; Molina, Silvia; Acuña-Amador, Luis et al. (2016) Deletion of IQGAP1 promotes Helicobacter pylori-induced gastric dysplasia in mice and acquisition of cancer stem cell properties in vitro. Oncotarget 7:80688-80699
Byrnes, Colleen; Lee, Y Terry; Meier, Emily R et al. (2016) Iron dose-dependent differentiation and enucleation of human erythroblasts in serum-free medium. J Tissue Eng Regen Med 10:E84-9

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