During the fiscal year, we accomplished the following: 1. Axl is a tyrosine kinase receptor that is important for hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival. Binding of growth arrest-specific protein 6 (Gas6) activates Axl signaling, but the mechanism of inactivation of the Axl receptor is poorly understood. We documented that IQGAP1 modulates Axl signaling. IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules. Our in vitro analysis revealed a direct interaction between the IQ domain of IQGAP1 and Axl. Analysis by both immunoprecipitation and proximity ligation assays demonstrated an association between Axl and IQGAP1 in cells and this interaction was decreased by Gas6. Unexpectedly, reducing IQGAP1 levels in cells significantly enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, IQGAP1 regulated the interaction of Axl with the epidermal growth factor receptor. Our data identify IQGAP1 as a previously undescribed suppressor of Axl and provide insight into regulation of Axl function. 2. The Ras family of small GTPases modulates numerous essential processes. Activating Ras mutations result in hyper-activation of selected signaling cascades, which leads to human diseases. The high frequency of Ras mutations in human malignant neoplasms has led to Ras being a desirable chemotherapeutic target. The IQGAP family of scaffold proteins binds to and regulates multiple signaling molecules, including the Rho family GTPases Rac1 and Cdc42. There are conflicting data in the published literature regarding interactions between IQGAP and Ras proteins. Initial reports showed no binding, but subsequent studies claim associations of IQGAP1 and IQGAP3 with K-Ras and H-Ras, respectively. Therefore, we set out to resolve this controversy. We demonstrated that neither endogenous IQGAP1 nor endogenous IQGAP3 binds to the major Ras isoforms, namely H-, K-, and N-Ras. Importantly, Ras activation by epidermal growth factor was not altered when IQGAP1 or IQGAP3 proteins are depleted from cells. Our data strongly suggest that IQGAP proteins are not functional interactors of H-, K-, or N-Ras and challenge the rationale for targeting the interaction of Ras with IQGAP for the development of therapeutic agents. 3. Transendothelial migration (TEM) of leukocytes across the endothelium is critical for inflammation. In the endothelium, TEM requires coordination of membrane movements and cytoskeletal interactions, including prominently, recruitment of the lateral border recycling compartment (LBRC). We recently identified the scaffold protein IQGAP1 in a screen for LBRC-interacting proteins. Knockdown of endothelial IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. Expression of truncated IQGAP1 constructs demonstrated that the calponin homology domain is required for IQGAP1 localization to endothelial borders and that the IQ domain, on the same IQGAP1 polypeptide, is required for its function in TEM. This is the first reported function of IQGAP1 requiring two domains to be present on the same polypeptide. Additionally, we demonstrated for the first time that IQGAP1 in endothelium is required for efficient TEM in vivo. These findings reveal a novel function for IQGAP1 and demonstrate that IQGAP1 in endothelial cells facilitates TEM by directing the LBRC to the site of TEM.

Agency
National Institute of Health (NIH)
Institute
Clinical Center (CLC)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIACL080017-09
Application #
10022066
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Clinical Center
Department
Type
DUNS #
City
State
Country
Zip Code
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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