关键词： IQGAP1   squamous cell carcinoma   inverted papilloma   tumor invasion   metastasis   Level of Evidence: NA  

摘要： Objectives/Hypothesis: The scaffold protein IQGAP1 is a ubiquitously expressed 190 kDa protein that participates in protein-protein interactions, integrating diverse signaling pathways. In this study, we examined the expression of IQGAP1 in sinonasal inverted papillomas (IP) and sinonasal squamous cell carcinomas (SCC), which include IP with SCC (IPcSCC) and SCC alone. We also tried to elucidate whether the amount of IQGAP1 expression is related with any clinicopathologic features of sinonasal SCCs. Study Design: This was a retrospective study. Methods: The expression of IQGAP1 was analyzed by means of immunohistochemistry. The degrees of IQGAP1 expression and subcellular localization (rim formation) were analyzed with respect to the following clinicopathologic variables: age, gender, T stage, histologic differentiation, local recurrence, and distant metastasis. Results: The expression of IQGAP1 was significantly higher in patients with IPcSCC (3.62 +/- 0.14), and SCC (2.93 +/- 0.34) compared with IP (1.43 +/- 0.26). The rim formation scale was significantly higher in IPcSCC (2.39 +/- 0.24) compared with IP (1.68 +/- 0.15). Distant metastasis in patients with SCC significantly correlated with an increased IQGAP1 rim formation (p = 0.011). Histologic differentiation was also significantly correlated with a positive staining scale for IQGAP1 in IPcSCCs (p = 0.038). Conclusion: These results indicated that IQGAP1 expression is associated with both the histologic differentiation of IPcSCCs and is present in SCC cases where distant metastasis has occurred.

关键词： CHD   Cytoskeletal scaffolding protein   Calcium-dependent interaction   Protein-protein crosslinking   ANS displacement assay  

摘要： IQGAPs are cytoskeletal scaffolding proteins which collect information from a variety of signalling pathways and pass it on to the microfilaments and microtubules. There is a well-characterised interaction between IQGAP and calmodulin through a series of IQ-motifs towards the middle of the primary sequence. However, it has been shown previously that the calponin homology domain (CHD), located at the N-terminus of the protein, can also interact weakly with calmodulin. Using a recombinant fragment of human IQGAP1 which encompasses the CHD, we have demonstrated that the CHD undergoes a calcium ion-dependent interaction with calmodulin. The CHD can also displace the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulphonate from calcium-calmodulin, suggesting that the interaction involves non-polar residues on the surface of calmodulin. Molecular modelling identified a possible site on the CHD for calmodulin interaction. The physiological significance of this interaction remains to be discovered.

关键词： Animals   Cell Cycle Proteins/genetics   Cell Cycle Proteins/metabolism   Cell Line, Tumor   Cell Proliferation   Cercopithecus aethiops   COS Cells   Gene Expression Regulation, Neoplastic   Humans   Models, Biological   ras GTPase-Activating Proteins/genetics   ras GTPase-Activating Proteins/metabolism   ras GTPase-Activating Proteins/physiology   rho GTP-Binding Proteins/genetics   rho GTP-Binding Proteins/metabolism   rho GTP-Binding Proteins/physiology   RNA Interference   Stomach Neoplasms/metabolism   Stomach Neoplasms/pathology  

摘要： Our previous research results showed that both Ras homolog family member C (RhoC) and IQ-domain GTPase-activating protein 1 (IQGAP1) were over-expressed in gastric cancer tissues and cells, but their role in tumorigenensis has not been addressed clearly. Herein we reported the proliferation stimulating effect of RhoC and IQGAP1 on gastric cancer cells and the interaction between two proteins in regulating the proliferation of gastric cancer cells. Plasmids and viral constructs encoding target siRNA and DNA were used to alter the expression of RhoC and IQGAP1. MTT method and BrdU incorporation assay were used for analyzing the effect of RhoC and different structures of IQGAP1 on proliferation. Protein levels of IQGAP1 and RhoC in cell lines were detected by Western blotting. Immunofluorescence and Co-Immunoprecipitation assays were applied to investigate the localization and binding between RhoC and IQGAP1. The results showed that RhoC, IQGAP1 and the C-terminal fragment of IQGAP1 significantly stimulated the proliferation of gastric cancer cells, and enhanced the expression of cyclin E and cyclin D1. By contrast, reduction of endogenous IQGAP1 or RhoC by siRNA attenuated cell proliferation. The depletion of IQGAP1 expression by siRNA significantly blocked the proliferative activity of constitutively active RhoC, while RhoC silencing by siRNA had no effect on IQGAP1-induced proliferation in gastric cancer cells. Co-immunoprecipitation and Immunofluorescence assays showed that RhoC and IQGAP1 bound each other. In conclusion, our results suggest that RhoC stimulates the proliferation of gastric cancer cells through recruiting IQGAP1 as an effector.

关键词： Breast Cancer   Cell Migration   Cell Proliferation   Protein-Protein Interactions   Rho GTPases   IQGAP1   Rho Effector   Rho Isoforms  

摘要： We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA(L63) and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA(L63)-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC(V14) mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.

关键词： Chlamydophila (Chlamydia) pneumoniae   VSMC   Cell adhesion   Cell migration   IQGAP1   ShRNA  

摘要： The mechanisms for Chlamydophila (Chlamydia) pneumoniae (C. pneumoniae) infection-induced atherosclerosis are still unclear. Cell adhesion has important roles in vascular smooth muscle cell (VSMC) migration required in the development of atherosclerosis. However, it is still unknown whether IQ domain GTPase-activating protein 1 (IQGAP1) plays pivotal roles in C. pneumoniae infection-induced the adhesion and migration of rat primary VSMCs. Accordingly, in this study, we demonstrated that rat primary VSMC adhesion (P < 0.001) and migration (P < 0.01) measured by cell adhesion assay and Transwell assay, respectively, were significantly enhanced after C. pneumoniae infection. Reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression levels of IQGAP1 in the infected rat primary VSMCs were found to increase gradually to reach a peak and then decrease gradually to a level similar to the control. We further showed that the increases in rat primary VSMC adhesion to Matrigel (P < 0.001) and migration (P < 0.01) caused by C. pneumoniae infection were markedly inhibited after IQGAP1 knockdown by a pool of four short hairpin RNAs. Taken together, our results suggest that C pneumoniae infection may promote the adhesion and migration of VSMCs possibly by upregulating the IQGAP1 expression. (c) 2012 Elsevier Ltd. All rights reserved.

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.

关键词： disruption   junctions   cells   Zonula Adherens   cadherin 5   Invasions   Escherichia coli   Actins   Carboxy-Terminal Amino Acid   Endothelial Cells  

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.