IQGAP1 in tumorigenesis

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Abstract

During the fiscal year, we accomplished the following: 1. IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. The only characterized functional phosphorylation sites in IQGAP1 are Ser-1441/Ser-1443, which we previously showed are catalyzed by the protein kinase C (PKC) isoforms PKCor PKC Phosphorylation of these amino acid residues promotes neurite outgrowth. By contrast, almost nothing was known about tyrosine phosphorylation. Therefore, we characterized and evaluated the function of IQGAP1 tyrosine phosphorylation. Using mass spectrometry-based analysis with our longstanding collaborator, Roland Annan, we identified four tyrosine residues, namely Tyr-172, Tyr-654, Tyr-855 and Tyr-1510, that are phosphorylated on IQGAP1 in human cells. Importantly under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines, IQGAP1 was phosphorylated exclusively on Tyr-1510. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src, or siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a non-phosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. (The construct is termed IQGAP1 Y1510A.) The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with wild-type IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Since increased MET signaling is implicated in the development and progression of several types of carcinomas, IQGAP1 may be a potential therapeutic target in selected malignancies. 2. Due to its fundamental role in energy control, AMP-activated protein kinase (AMPK) has been the focus of intense research over the last few years. A large body of evidence supports the concept that activation of AMPK is beneficial for the prevention and treatment of a variety of chronic diseases, particularly metabolic disorders, such as type 2 diabetes mellitus and the metabolic syndrome. We previously documented that the scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase (MAPK) and AKT signaling cascades. IQGAP1 was implicated in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. We demonstrated a previously unidentified direct interaction of IQGAP1 with both AMPK and Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2), an upstream activator of AMPK. Both CaMKK2 and AMPK associate with IQGAP1 in cells. Moreover, activation of AMPK is reduced in cells lacking IQGAP1. Importantly, transcriptional regulation of some gluconeogenesis and fatty acid synthesis genes is impaired in IQGAP1-null mice. Combined with our data that IQGAP1 is necessary for optimal activation of insulin signaling, the data in this study raise the possibility that the interaction between AMPK and IQGAP1 could be a potential target for the development of new therapeutic agents for metabolic disorders. 3. The mitogen-activated protein kinase (MAPK) cascade is a fundamental signaling pathway that regulates cell fate decisions in response to external stimuli. Several scaffold proteins bind directly to kinase components of this pathway and regulate their activation by growth factors. One of the best studied MAPK scaffolds is kinase suppressor of Ras1 (KSR1). While several different investigators have shown that Ca2+ modulates MAPK signaling, the molecular mechanisms by which this occurs are incompletely understood. In this study, we showed for the first time that Ca2+ /calmodulin, but not apo-calmodulin, binds directly to KSR1. Moreover, endogenous calmodulin and KSR1 co-immunoprecipitate from mammalian cell lysates in a Ca2+-dependent manner. The cell-permeable calmodulin antagonist CGS9343B impaired the ability of epidermal growth factor (EGF) to activate MAPK signaling. Investigation of the molecular mechanism revealed that CGS9343B reduces both the translocation of KSR1 to the plasma membrane and the formation of KSR1-ERK (extracellular-regulated kinase) complexes elicited by EGF. In this study we describe for the first time an intersection between the Ca2+ signaling and MAPK pathways via a direct interaction between Ca2+/calmodulin and KSR1. These observations expand our comprehension of essential intracellular signaling networks. Moreover, since normal MAPK homeostasis is disrupted in several human diseases, ranging from cancer and developmental disorders to Alzheimers disease and age-related macular degeneration, our findings provide an additional avenue of exploration to develop new therapeutic approaches to ameliorate these conditions. 4. In 2016 we identified that IQGAP1 participates in Hippo signaling. Hippo regulates a complex network which mediates several biological functions, ranging from cell proliferation to apoptosis. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. We documented that IQGAP1 binds YAP and modulates its transcriptional activity. Since IQGAP1 scaffolds several signaling cascades, we set out to investigate whether it functions as a scaffold in the Hippo pathway. YAP is regulated by the kinase module of the Hippo pathway, which includes large tumor suppressor 1 and 2 (LATS1/2) and mammalian STE20-like protein kinase 1 and 2 (MST1/2). In collaboration with David Matallanas, we showed that IQGAP1 co-immunoprecipitates with MST2 and LATS1 from cultured mammalian cells. Using a panel of deletion mutants of selected regions of IQGAP1, we observed that both MST2 and LAT1 bind to the IQ domain of IQGAP1. Our results indicate that IQGAP1 scaffolds MST2 and LATS1 and suppresses their kinase activity, thereby influencing YAP-dependent transcription. Additionally, we observed that IQGAP1 is a negative regulator of the non-canonical pro-apoptotic pathway and may enable the crosstalk between this pathway and the ERK and AKT signalling modules. Moreover, our data show that bile acids regulate the IQGAP1-MST2-LATS1 signalling module in hepatocellular carcinoma cells, which could be necessary for the inhibition of MST2-dependent apoptosis and hepatocyte transformation. Collectively, these findings provide additional insight into the mechanisms by which IQGAP1 functions as an oncogene in hepatocellular carcinoma. 5. Age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Loss of visual acuity in neovascular age-related macular degeneration occurs rapidly when factors activate choroidal endothelial cells to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization. Active Rac1 is required for migration of choroidal endothelial cell and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. In collaboration with Mary Elizabeth Hartnett, we addressed the hypothesis that IQGAP1, which binds Rac1, regulates pathologic Rac1 in choroidal endothelial cell migration and choroidal neovascularization. Compared to littermate Iqgap1+/+ mice, Iqgap1/ mice had reduced volumes of laser-induced choroidal neovascularization as well as decreased active Rac1 and phosphorylated VEGF receptor 2 (VEGFR2) within lectin-stained choroidal n