关键词： Shigella flexneri   Shigellosis   Cell proliferation   Transfection   Phosphorylation   Small interfering RNA   HeLa cells   Actins  

摘要： 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 demonstrate 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 show 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 demonstrate 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.

关键词： actin   cell migration   membrane   microtubule   microtubule dynamics   scaffold protein  

摘要： Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.

关键词： Actin   Calcium-binding protein   Cell cortex   EF hand   Membrane  

摘要： The cortical cytoskeleton constitutes an important subcellular structure that determines cell shape and regulates cell migration as well as membrane traffic to and from the plasma membrane. Many components of the cortical cytoskeleton have been identified including structural and scaffolding proteins, membrane-cytoskeleton linker proteins and signaling intermediates. We describe here an association of the membrane-F-actin linker protein ezrin with the scaffolding protein IQGAP1 that serves as a hub for concentrating different signaling complexes. Both, ezrin and IQGAP1 bind in a Ca2+-dependent manner to the EF hand protein S100P and complexes consisting of Ca2+-bound S100P, IQGAP1 and ezrin can be isolated by immunoprecipitation. Ezrin and IQGAP1 also interact in the absence of Ca2+, thus independent of S100P. Direct ezrin-IQGAP1 interaction can be shown with the purified proteins. It is mediated via the N-terminal FERM domain of ezrin and the IQ domain of IQGAP1, respectively. Ezrin and IQGAP1 colocalize in the submembraneous cytoskeleton and in cellular protrusions of human epithelial cells and knockdown of ezrin reduces the cortical localization of IQGAP1. Thus, ezrin appears to participate in recruiting IQGAP1 to the cell cortex thereby establishing a close connection between membrane-F-actin contacts and actin regulators that can be assembled by IQGAP1. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. (C) 2014 Elsevier B.V. All rights reserved.

关键词： Actin   Calcium-binding protein   Cell cortex   EF hand   Membrane  

摘要： The cortical cytoskeleton constitutes an important subcellular structure that determines cell shape and regulates cell migration as well as membrane traffic to and from the plasma membrane. Many components of the cortical cytoskeleton have been identified including structural and scaffolding proteins, membrane–cytoskeleton linker proteins and signaling intermediates. We describe here an association of the membrane-F-actin linker protein ezrin with the scaffolding protein IQGAP1 that serves as a hub for concentrating different signaling complexes. Both, ezrin and IQGAP1 bind in a Ca 2 + -dependent manner to the EF hand protein S100P and complexes consisting of Ca 2 + -bound S100P, IQGAP1 and ezrin can be isolated by immunoprecipitation. Ezrin and IQGAP1 also interact in the absence of Ca 2 + , thus independent of S100P. Direct ezrin–IQGAP1 interaction can be shown with the purified proteins. It is mediated via the N-terminal FERM domain of ezrin and the IQ domain of IQGAP1, respectively. Ezrin and IQGAP1 colocalize in the submembraneous cytoskeleton and in cellular protrusions of human epithelial cells and knockdown of ezrin reduces the cortical localization of IQGAP1. Thus, ezrin appears to participate in recruiting IQGAP1 to the cell cortex thereby establishing a close connection between membrane-F-actin contacts and actin regulators that can be assembled by IQGAP1. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. [ABSTRACT FROM AUTHOR]

关键词： cDNA   complementary DNA   IQGAP   IQ motif–containing-GTPase activating protein   MAPK   mitogen-activated protein kinase   pERK   phospho ERK   SCC   squamous cell carcinoma  

摘要： IQ motif-containing GTPase-activating protein (IQGAP) scaffolding proteins regulate many essential cellular processes including growth factor receptor signaling, cytoskeletal rearrangement, adhesion, and proliferation and are highly expressed in many cancers. Using genetically engineered human skin tissue in vivo, we demonstrate that diminished, sub-physiologic expression of IQGAP1 or IQGAP3 is sufficient to maintain normal epidermal homeostasis, whereas significantly higher levels are required to support tumorigenesis. To target this tumor-specific IQGAP requirement in vivo, we engineered epidermal keratinocytes to express individual IQGAP protein domains designed to compete with endogenous IQGAPs for effector protein binding. Expression of the IQGAP1-IQ motif decoy domain in epidermal tissue in vivo inhibits oncogenic Ras-driven mitogen-activated protein kinase signaling and antagonizes tumorigenesis, without disrupting normal epidermal proliferation or differentiation. These findings define essential non-redundant roles for IQGAP1 and IQGAP3 in the epidermis and demonstrate the potential of IQGAP antagonism for cancer therapy.

关键词： Hepatocellular carcinoma   Transfection   Small interfering RNA   Cell migration   Cell proliferation   Hyperexpression techniques   Differentiated tumors   Metastasis  

摘要： The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with beta-catenin and regulates beta-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and beta-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and beta-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease beta-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and beta-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and beta-catenin contribute to HCC progression by promoting cell proliferation and migration.

关键词： Extracellular vesicles   Liquid chromatography-tandem mass spectrometry   Colorectal cancer   Proteomics   14-3-3   STAT1   IQGAP1   Raf-1   prohibitin   NF-kappa B   Luciferase reporter assay  

摘要： Background: Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. Methods: EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-kappa B. Results: This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-kappa B which was inhibited by the NF-kappa B inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Conclusions: Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

摘要： IQ motif containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell derived factor-1 (SDF-1;also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1(+) endosomes, and decreased efficiency of CXCR4 recycling. SDF-1 induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to a-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1(+) endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1(+) endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types.

关键词： Scaffold proteins   Lymphocytes   Signaling   IQGAP1  

摘要： The intracellular spatiotemporal organization of signaling events is critical for normal cellular function. In response to environmental stimuli, cells utilize highly organized signaling pathways that are subject to multiple layers of regulation. However, the molecular mechanisms that coordinate these complex processes remain an enigma. Scaffolding proteins (scaffolins) have emerged as critical regulators of signaling pathways, many of which have well-described functions in immune cells. IQGAP1, a highly conserved cytoplasmic scaffold protein, is able to curb, compartmentalize, and coordinate multiple signaling pathways in a variety of cell types. IQGAP1 plays a central role in cell-cell interaction, cell adherence, and movement via actin/tubulin-based cytoskeletal reorganization. Evidence also implicates IQGAP1 as an essential regulator of the MAPK and Wnt/beta-catenin signaling pathways. Here, we summarize the recent advances on the cellular and molecular biology of IQGAP1. We also describe how this pleiotropic scaffolin acts as a true molecular puppeteer, and highlight the significance of future research regarding the role of IQGAP1 in immune cells. (C) 2015 Elsevier Ltd. All rights reserved.

关键词： FRET: Forster (or fluorescence) resonance energy transfer   E%: energy-transfer efficiency   quantitative FRET analysis   actin nucleation   N-WASP   Rac1   Cdc42   IQGAP1   ROI: region of interest   PFRET: processed FRET or spectral bleedthrough-corrected FRET  

摘要： Experiments using live cell 3-color Forster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. (c) 2015 International Society for Advancement of Cytometry