关键词： p120   NF-κB   IL-8   炎症反应   气道上皮细胞   CSE   IQGAP1   β-catenin   细胞粘附   气道上皮细胞  

摘要： 实验背景 气道上皮是呼吸系统对抗有害刺激的第一道防线,各种病原微生物在机体抵抗力低下时均可引起肺部感染性疾病。其中以革兰阴性菌细胞壁的主要成分脂多糖(lipopolysaccharide, LPS)为主要致病因素,不仅引起肺部炎症反应,还可使原有肺部疾病发展和加重。气道上皮在致病因素的攻击下受到损伤,随后启动自身修复机制。LPS致气道上皮细胞损伤后,可促使细胞发生坏死、凋亡和分泌前炎症因子等反应,这些反应共同推动肺损伤的发展过程。大量研究证明,核因子-κB (nuclear factor, NF-κB)信号途径参与调节细胞生长、分化、增殖、存活、凋亡及炎症反应过程中多种基因的转录,与LPS引起的肺损伤密切相关。然而,LPS导致肺部炎症反应的分子机制还远未阐明。 NF-κB在正常情况下与其抑制蛋白ⅠκB结合以无活性的形式存在于静息细胞胞浆中。外界刺激可引起ⅠκB被迅速磷酸化、泛素化而降解,从而释放NF-κB活性亚单位转位入核,激活下游靶基因转录。P120-catenin(p120)是连环素家族成员,可在细胞膜上与上皮钙粘蛋白(E-Cadherin, E-Cad)相互结合而调节细胞粘附。近年来有研究表明,p120通过调节NF-κB活性参与表皮炎症反应。我们推测在气道炎症反应中,p120也可能通过NF-κB信号途径发挥调节作用。 实验目的 本实验使用脂多糖刺激建立体外气道炎症反应模型,初步探讨脂多糖刺激后气道上皮细胞中p120的表达变化及其对NF-κB信号通路的影响,从而进一步分析气道炎症发生发展的机制。 实验方法 使用脂多糖刺激建立体外气道炎症反应模型；采用免疫荧光共聚焦成像、Western blot、核浆分离方法检测p120、NF-κB、IκBα等蛋白表达及定位变化；采用荧光定量PCR、定量酶联免疫吸附实验(ELISA)检测IL-8表达变化；采用荧光素酶报告基因分析的方法检测NF-κB的活性；通过瞬时转染及小干扰RNA技术改变细胞中p120的表达量再检测p120对NF-κB信号途径的影响。 实验结果 (1)细胞瞬时转染NF-κB报告质粒后,报告基因分析结果显示,LPS刺激可引起气道上皮细胞中NF-κB信号的活化；同时荧光定量PCR和ELISA结果显示NF-κB靶基因IL-8--一种前炎症因子的表达亦有明显增多。 (2)p120在正常气道上皮细胞中含量丰富,而LPS刺激后,其表达迅速下调,早在15 min时即可表现出来。基于以上两点,我们推测在LPS所致气道炎症反应中,p120与NF-κB信号途径可能存在某种联系。接下来,我们先探讨NF-κB信号活化的机制。 (3)实验发现,在LPS刺激15 min-30 min时,NF-κB的抑制蛋白IκBα迅速降解至几近消失；其磷酸化形式在正常细胞中几乎检测不到,但在LPS处理15 min-30 min这段时间内含量显著增加,在IκBα恢复正常水平后,p-IKBa又再次消失不见。 (4)核浆分离试验及免疫荧光共聚焦结果均显示LPS促使NF-κB活性亚单位p65发生核内转位。 结果(3)和(4)表明,LPS在气道上皮细胞中引起的NF-κB活化是通过使IκBa发生磷酸化降解,从而导致p65由NF-κB/IκB复合体中释放并转位入核激活相应靶基因转录。接下来,我们通过对细胞转染p120质粒或小干扰RNA,研究p120对NF-κB信号活化的影响。 (5)实验结果显示,过表达p120可以部分抑制NF-κB的活性及IL-8的表达,但免疫荧光及核浆分离结果依然可以观测到p65核转位。 (6)小干扰RNA敲低p120的表达后,免疫荧光结果显示,LPS可引起p65更强烈的核染色；同时核浆分离产物的免疫印迹结果也证实阻断p120表达可进一步促进LPS诱导的p65核转位。 (7)阻断p120的表达可大大提升NF-κB的转录活性及其靶基因IL-8的表达。 实验结论 本实验表明： (1)LPS可导致气道上皮细胞中p120表达下调。 (2)LPS可诱导气道上皮细胞NF-κB信号的活化及其靶基因IL-8的表达。 (3) NF-κB信号途径的活化是通过IκBα的磷酸化降解释放p65入核实现的。 (4)过表达p120可部分抑制NF-κB信号的活化；敲低p120能显著促进NF-κB信号的活化。 本实验提示： 在LPS引起的气道炎症反应中,p120可能负性调节NF-κB信号途径的活化, 从而抑制气道炎症反应。 实验背景 大量研究表明,吸烟可从多个方面损伤气道上皮细胞,更是导致

关键词： IQGAP1   Iqg1p   mTOR   Cdc42   exocyst   protein trafficking   cell proliferation  

摘要： IQGAP1, an effector of CDC42p GTPase, is a widely conserved, multifunctional protein that bundles F-actin through its N-terminus and binds microtubules through its C-terminus to modulate the cell architecture. It has emerged as a potential oncogene associated with diverse human cancers. Therefore, IQGAP1 has been heavily investigated;regardless, its precise cellular function remains unclear. Work from yeast suggests that IQGAP1 plays an important role in directed cell growth, which is a conserved feature crucial to morphogenesis, division axis, and body plan determination. New evidence suggests a conserved role for IQGAP1 in protein synthesis and membrane traffic, which may help to explain the diversity of its cellular functions. Membrane traffic mediates infections by intracellular pathogens and a range of degenerative human diseases arise from dysfunctions in intracellular traffic;thus, elucidating the mechanisms of cellular traffic will be important in order to understand the basis of a wide range of inherited and acquired human diseases. Recent evidence suggests that IQGAP1 plays its role in cell growth through regulating the conserved mTOR pathway. The mTOR signaling cascade has been implicated in membrane traffic and is activated in nearly all human cancers, but clinical response to the mTOR-specific inhibitor rapamycin has been disappointing. Thus, understanding the regulators of this pathway will be crucial in order to identify predictors of rapamycin sensitivity. In this review, I discuss emerging evidence that supports a potential role of IQGAP1 in regulating membrane traffic via regulating the mTOR pathway.

关键词： IQGAP1   Beta-catenin   Cancer   Cell adhesion   Cell migration  

摘要： IQGAP1 is a key mediator of several distinct cellular processes, in particular cytoskeletal rearrangements. Recent studies have implicated a potential role for IQGAP1 in cancer, supported by the over-expression and distinct membrane localisation of IQGAP1 observed in a range of tumours. IQGAP1 is thought to contribute to the transformed cancer cell phenotype by regulating signalling pathways involved in cell proliferation and transformation, weakening of cell:cell adhesion contacts and stimulation of cell motility and invasion. In this review we discuss these different functional and regulatory roles of IQGAP1 and its homologues in relation to their potential impact on tumourigenesis. (C) 2009 Elsevier Inc. All rights reserved.

关键词： Adherens Junction   Rac1 Activity   P100 Fraction   Rac1 Inhibitor   Coimmunoprecipitation Experiment  

摘要： Background: Monomeric GTPases of the Rho family control a variety of cellular functions including actin cytoskeleton organisation, cell migration and cell adhesion. Defects in these regulatory processes are involved in tumour progression and metastasis. The development of metastatic carcinoma is accompanied by deregulation of adherens junctions, which are composed of E-cadherin/beta- and alpha-catenin complexes. Results: Here, we show that the activity of the monomeric GTPase Rac1 contributes to inhibition of E-cadherin-mediated cell-cell adhesion in pancreatic carcinoma cells. Stable expression of constitutively active Rac1(V12) reduced the amount of E-cadherin on protein level in PANC-1 pancreatic carcinoma cells, whereas expression of dominant negative Rac1(N17) resulted in an increased amount of E-cadherin. Extraction of proteins associated with the actin cytoskeleton as well as coimmunoprecipitation analyses demonstrated markedly decreased amounts of E-cadherin/catenin complexes in Rac1(V12)-expressing cells, but increased amounts of functional E-cadherin/catenin complexes in cells expressing Rac1(N17). Cell aggregation and migration assays revealed, that cells containing less E-cadherin due to expression of Rac1(V12), exhibited reduced cell-cell adhesion and increased cell motility. The Rac/Cdc42 effector protein IQGAP1 has been implicated in regulating cell-cell adhesion. Coimmunoprecipitation studies showed a decrease in the association between IQGAP1 and beta-catenin in Rac1(V12)-expressing PANC-1 cells and an association of IQGAP1 with Rac1(V12). Elevated association of IQGAP1 with the E-cadherin adhesion complex via beta-catenin correlated with increased intercellular adhesion of PANC-1 cells. Conclusion: These results indicate that active Rac1 destabilises E-cadherin-mediated cell-cell adhesion in pancreatic carcinoma cells by interacting with IQGAP1 which is associated with a disassembly of E-cadherin-mediated adherens junctions. Inhibition of Rac1 acti

关键词： GSK-3   IQGAP1   CLASP2   EB1   Microtubule  

摘要： Polarised cell migration is required for various cell behaviours and functions. Actin and microtubules are coupled structurally and distributed asymmetrically along the front-rear axis of migrating cells. CLIP-associating proteins (CLASPs) accumulate near the ends of microtubules at the front of migrating cells to control microtubule dynamics and cytoskeletal coupling. Regional inhibition of GSK-3 beta is responsible for this asymmetric distribution of CLASPs. However, it is not known how GSK-3 beta regulates the activity of CLASPs for linkage between actin and microtubules. Here we identified IQGAP1, an actin-binding protein, as a novel CLASP-binding protein. GSK-3 beta directly phosphorylates CLASP2 at Ser533 and Ser537 within the region responsible for the IQGAP1 binding. Phosphorylation of CLASP2 results in the dissociation of CLASP2 from IQGAP1, EB1 and microtubules. At the leading edges of migrating fibroblasts, CLASP2 near microtubule ends partially colocalises with IQGAP1. Expression of active GSK-3 beta abrogates the distribution of CLASP2 on microtubules, but not that of a nonphosphorylatable CLASP2 mutant. The phosphorylated CLASP2 does not accumulate near the ends of microtubules at the leading edges. Thus, phosphorylation of CLASP2 by GSK-3 beta appears to control the regional linkage of microtubules to actin filaments through IQGAP1 for cell migration.

关键词： Cancer   IQGAP1   IQGAP2   IQGAP3   Metastasis   Neoplasia   Tumorigenesis  

摘要： The IQGAP family comprises three proteins in humans. The best characterized is IQGAP1, which participates in protein-protein interactions and integrates diverse signaling pathways. IQGAP2 and IQGAP3 harbor all the domains identified in IQGAP1, but their biological roles are poorly defined. Proteins that bind IQGAP1 include Cdc42 and Rac1, E-cadherin, beta-catenin, calmodulin and components of the mitogen-activated protein kinase pathway, all of which are involved in cancer. Here, we summarize the biological functions of IQGAPs that may contribute to neoplasia. Additionally, we review published data which implicate IQGAPs in cancer and tumorigenesis. The cumulative evidence suggests IQGAP1 is an oncogene while IQGAP2 may be a tumor suppressor. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

关键词： Cell growth   IQGAP1   Protein synthesis  

摘要： Cell proliferation requires close coordination of cell growth and division to ensure constant cell size through the division cycles. IQGAP1, an effector of CDC42 GTPase has been implicated in the modulation of cell architecture, regulation of exocytosis and in human cancers. The precise mechanism underlying these activities is unclear. Here, we show that IQGAP1 regulates cell proliferation, which requires phosphorylation of IQGAP1 and binding to CDC42. Expression of the C-terminal region of IQGAP1 enhanced cellular transformation and migration, but reduced the cell size, whereas expression of the N-terminus increased the cell size, but inhibited cell transformation and migration. The N-terminus of IQGAP1 interacts with mTOR, which is required for IQGAP1-mediated cell proliferation. These findings are consistent with a model where IQGAP1 serves as a phosphorylation-sensitive conformation switch to regulate the coupling of cell growth and division through a novel CDC42-mTOR pathway, dysregulation of which generates cellular transformation.

关键词： HYDROLYSIS   PROTEINS -- Analysis   AMINO acid sequence   PROTEINS -- Affinity labeling   VOLUMETRIC analysis  

摘要： IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic "arginine finger" seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099-1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42 center dot GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.

关键词： PHOSPHOLIPASES   HYPEROXIA   PULMONARY artery   BUTANOL   PHOSPHORYLATION   TYROSINE  

摘要： Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47(phox) to cell periphery, and ROS production. Exposure of [P-32] orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O-2 and 5% CO2) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [P-32] phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O-2(radical anion)/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [P-32] phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47(phox) redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47(phox) translocation and ROS formation in human lung endothelial cells.