关键词： Caco-2 cells   Cell migration   Phosphorylation   Actins   Epithelial cells   Cell membranes   Diluents   Lactones  

摘要： Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxododecanoyl- L-homoserine lactone 3O-C-12-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C-12-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C-12-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C-12-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C-12-HSL with a sensor bioassay. Moreover, 3O-C-12-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P. aeruginosa 3O-C-12-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C-12-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial - human cell communication.

关键词： CDC42   Cytoskeleton   Protein   phosphorylation   Cancer-associated   mutation   Protein-protein   interaction  

摘要： AIM:To understand the interaction of human IQGAP1 and CDC42,especially the effects of phosphorylation and a cancer-associated mutation. METHODS:Recombinant CDC42 and a novel C-termi- nal fragment of IQGAP1 were expressed in,and puri- fied from,Escherichia *** directed mutagenesis was used to create coding sequences for three phos- phomimicking variants(S1441E,S1443D and S1441E/ S1443D)and to recapitulate a cancer-associated mu- tation(M1231I).These variant proteins were also ex- pressed and ***-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and *** interactions were quanti- fied using surface plasmon resonance *** modelling was employed to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant. RESULTS:The novel,C-terminal region of human IQGAP1 (residues 877-1558)is soluble following expression and *** is also capable of binding to CDC42,as judged by crosslinking *** appears to be strongest in the presence of added *** three phosphomimicking mutants had different affini- ties for CDC42.S1441E had an approximately 200-fold reduction in affinity compared to wild *** was caused largely by a dramatic reduction in the associa- tion rate *** contrast,both S1443D and the double variant S1441E/S1443D had similar affinities to the wild *** cancer-associated variant,M1231I, also had a similar affinity to wild ***,in the case of this variant,both the association and dis- sociation rate constants were reduced approximately *** modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region,revealed no gross structural changes compared to wild type(root mean square deviation of 0.564over 5556 equivalent atoms).However,pre- dictions of the flexibility o

关键词： Hyaluronan   CD44   IQGAP1   Cell migration   Cell proliferation   Rho-family GTPases  

摘要： IQGAP1, an essential scaffolding protein, forms a complex with the hyaluronan receptor CD44. In this study, we have examined the importance of IQGAP1 for hyaluronan-mediated fibroblast migration and proliferation. Hyaluronan induced formation of F-actin fibers and focal adhesions, which was dependent on IQGAP1. IQGAP1 was required for hyaluronan- but not for platelet-derived growth factor (PDGF)-BB-induced cell migration, and was required for both hyaluronan- and PDGF-BB-mediated fibroblast proliferation, but not for proliferation induced by 10% fetal bovine serum. Depletion of IQGAP1 suppressed hyaluronan-induced activation of Rac1 and enhanced the activation of RhoA. Taken together, these findings indicate important roles for IQGAP1 in hyaluronan-stimulated migration and proliferation of fibroblasts. (C) 2012 Elsevier Inc. All rights reserved.

摘要： The transcellular entry of Escherichia coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain oedema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E.?coli K1 interacts with its receptor, Ecgp96, to induce PKC-a phosphorylation, adherens junction (AJ) disassembly (by dislodging beta-catenin from VE-cadherin), and remodelling of actin in HBMEC. We report here that IQGAP1 mediates beta-catenin dissociation from AJs to promote actin polymerization required for E.?coli K1 invasion of HBMEC. Overexpression of C-terminal truncated IQGAP1 (IQ?C) that cannot bind beta-catenin prevents both AJ disruption and E.?coli K1 entry. Of note, phospho-PKC-a interacts with the C-terminal portion of Ecgp96 as well as with VE-cadherin after IQGAP1-mediated AJ disassembly. HBMEC overexpressing either C-terminal truncated Ecgp96 (Ecgp96?200) or IQ?C upon infection with E.?coli showed no interaction ofphospho-PKC-a with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC-a phosphorylation and association of phospho-PKC-a with Ecgp96, and then signals IQGAP1 to detach beta-catenin from AJs. Subsequently, IQGAP1/beta-catenin bound actin translocates to the site of E.?coli K1 attachment to promote invasion.

关键词： S1P   cortical actin   proteomics  

摘要： Bhattacharya M, Su G, Su X, Oses-Prieto JA, Li JT, Huang X, Hernandez H, Atakilit A, Burlingame AL, Matthay MA, Sheppard D. IQGAP1 is necessary for pulmonary vascular barrier protection in murine acute lung injury and pneumonia. Am J Physiol Lung Cell Mol Physiol 303: L12-L19, 2012. First published May 4, 2012;doi:10.1152/ajplung.00375.2011.-We recently reported that integrin alpha v beta(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel beta(3)-integrin binding partner IQGAP1. Like integrin beta(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin alpha v beta(3) to the cell-cell junction. Similar to beta(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of I-125-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin alpha v beta(3).

摘要： The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8(+) T cells. However, IQGAP1-deficient CD8(+) T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-gamma production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms. The Journal of Immunology, 2012, 188: 6135-6144.

关键词： Small interfering RNA   Transfection   Cell differentiation   Kidneys   Cytoskeleton   Thoracic diaphragm   Protein interactions   Actins  

摘要： IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (alpha-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.

关键词： IQGAP1   Aurora-A   Protein degradation   Tumorigenesis  

摘要： IQGAP1, a ubiquitously expressed scaffold protein, has been identified in a wide range of organisms. It participates in multiple aspects of cellular events by binding to and regulating numerous interacting proteins. In our present study, we identified a new IQGAP1 binding protein named Aurora-A which is an oncogenic protein and overexpressed in various types of human tumors. In vitro analysis with GST-Aurora-A fusion proteins showed a physical interaction between Aurora-A and IQGAP1. Moreover, the binding also occurred in HeLa cells as endogenous Aurora-A co-immunoprecipitated with IQGAP1 from the cell lysates. Overexpression of IQGAP1 resulted in an elevation of both expression and activity of Aurora-A kinase. Endogenous IQGAP1 knockdown by siRNA promoted Aurora-A degradation whereas IQGAP1 overexpression enhanced the stability of Aurora-A. Additionally, we documented that the IQGAP1-induced cell proliferation was suppressed by knocking down Aurora-A expression. Taken together, our results showed an unidentified relationship between Aurora-A and IQGAP1, and provided a new insight into the molecular mechanism by which IQGAP1 played a regulatory role in cancer. (C) 2012 Elsevier Inc. All rights reserved.

关键词： IQGAP1   Proliferation   Secretion   mTOR  

摘要： Defining the mechanisms that control cell growth and division is crucial to understanding cell homeostasis, which impacts human diseases such as cancer and diabetes. IQGAP1, a widely conserved effector and/or regulator of the GTPase CDC42, is a putative oncoprotein that controls cell proliferation;however, its mechanism in tumorigenesis is unknown. The mechanistic target of rapamycin (mTOR) pathway, the center of cell growth control, is commonly activated in human cancers, but has proved to be an ineffective clinical target because of an incomplete understanding of its mechanisms in cell growth inhibition. Using complementary studies in yeast and mammalian cells, we examined a potential role for IQGAP1 in regulating the negative feedback loop (NFL) of mTOR complex 1 (mTORC1) that controls cell growth. Two-hybrid screens identified the yeast TORC1-specific subunit Tco89p as an Iqg1p-binding partner, sharing roles in rapamycin-sensitive growth, axial-bud-site selection and cytokinesis, thus coupling cell growth and division. Mammalian IQGAP1 binds mTORC1 and Akt1 and in response to epidermal growth factor (EGF), cells expressing the mTORC1-Akt1-binding region (IQGAP1(IR-WW)) contained attenuated phosphorylated ERK1/2 (ERK1/2-P) activity and inactive glycogen synthase kinase 3 alpha/beta (GSK3 alpha/beta), which control apoptosis. Interestingly, these cells displayed a high level of Akt1 S473-P, but an attenuated level of the mTORC1-dependent kinase S6K1 T389-P and induced mTORC1-Akt1- and EGF-dependent transformed phenotypes. Moreover, IQGAP1 appears to influence cell abscission and its activity is elevated in carcinoma cell lines. These findings support the hypothesis that IQGAP1 acts upstream on the mTORC1-S6K1 -> Akt1 NFL and downstream of it, to couple cell growth and division, and thus like a rheostat, regulates cell homeostasis, dysregulation of which leads to tumorigenesis or other diseases. These results could have implications for the development of the next