关键词： ErbB2   IQGAP1   lamellipodia   PTB domain   ShcA  

摘要： Adaptor proteins respond to stimuli and recruit downstream complexes using interactions conferred by associated protein domains and linear motifs. The ShcA adaptor contains two phosphotyrosine recognition modules responsible for binding activated receptors, resulting in the subsequent recruitment of Grb2 and activation of Ras/MAPK. However, there is evidence that Grb2-independent signalling from ShcA has an important role in development. Using mass spectrometry, we identified the multidomain scaffold IQGAP1 as a ShcA-interacting protein. IQGAP1 and ShcA co-precipitate and are co-recruited to membrane ruffles induced by activated receptors of the ErbB family, and a reduction in ShcA protein levels inhibits the formation of lamellipodia. We used NMR to characterize a direct, non-canonical ShcA PTB domain interaction with a helical fragment from the IQGAP1 N-terminal region that is pTyr-independent. This interaction is mutually exclusive with binding to a more conventional PTB domain peptide ligand from PTP-PEST. ShcA-mediated recruitment of IQGAP1 may have an important role in cytoskeletal reorganization downstream of activated receptors at the cell surface. The EMBO Journal (2010) 29, 884 -896. doi: 10.1038/emboj.2009.399;Published online 14 January 2010

关键词： 支架蛋白   肿瘤   GTPase   蛋白相互作用   哺乳动物   催化域   同系物   全身性  

摘要： IQGAPs(IQ motif containing GTPase activating proteins)是近年新发现的一个蛋白家族,其序列中含有类似于RasGAPs催化域的广泛序列和位于N端的4个可与钙调蛋白相互作用的IQ模体,在哺乳动物中有3个同源物IQGAP 1～***1是3个人类IQGAP同系物中发现最早的1个~([1]),不同于IQGAP2和IQGAP3限制性地表达于肝和肠道等器官,其呈现出一种全身性地表达~([2-3]).

摘要： To identify key genes involved in the complex multistep process of hepatotumorigenesis, we reduced multivariate clinicopathological variables by using the Long-Evans Cinnamon rat, a model with naturally occurring and oxidative stress-induced hepatotumorigenesis. Gene expression patterns were analyzed serially by profiling liver tissues from rats of a naive status (4 weeks old), through to those with chronic hepatitis (26 and 39 weeks old) to tumor development (67 weeks old). Of 31 099 probe sets used for microarray analysis, 87 were identified as being upregulated in a stepwise manner during disease progression and tumor development. Quantitative real-time reverse transcription-polymerase chain reaction and statistical analyses verified that IQGAP1 and vimentin mRNA expression levels increased significantly throughout hepatotumorigenesis. A hierarchical clustering algorithm showed both genes clustered together and in the same cluster group. Immunohistochemical and western blot analyses showed similar increases in protein levels of IAGAP1 and vimentin. Finally, pathway analyses using text-mining technology with more comprehensive and recent gene-gene interaction data identified IQGAP1 and vimentin as important nodes in underlying gene regulatory networks. These findings enhance our understanding of the multistep hepatotumorigenesis and identification of target molecules for novel treatments.

关键词： Adherens junctions   Cytoskeleton   IQGAP1  

摘要： Leukocyte movement from the blood to the tissues is a fundamental process in acute and chronic inflammatory diseases. While the role of endothelial cells (EC) to recruit leukocytes to sites of inflammation is well known, the mechanisms that control remodeling of EC shape and adhesive contacts during leukocyte transendothelial migration (TEM) are not completely understood. We studied the role of IQGAP1, an adaptor protein that binds to filamentous-actin and microtubules (MT) at interendothelial junctions, during lymphocyte TEM. EC IQGAP1 knockdown decreases MT tethered to the adherens junction, and decreases lymphocyte TEM to ∼70% ( p <0.05) versus control. Similarly, loss of adherens junction-associated MT induced by brief nocodazole (ND) treatment decreases lymphocyte TEM to ∼65% of control ( p <0.01). Confocal microscopy imaging indicates that EC IQGAP1 knockdown and MT depolymerization both result in lymphocyte accumulation above the vascular endothelial cadherin (VE-cadherin) junctions and reduces the fraction of adherent lymphocytes that complete diapedesis across interendothelial cell junctions. However, we observe no change in VE-cadherin gap formation underlying adherent lymphocytes among control, IQGAP1 knockdown, or MT depolymerised EC monolayers. These data indicate that IQGAP1 contributes to MT stability at endothelial junctions. Further, IQGAP1 is involved in junction remodeling required for efficient lymphocyte diapedesis, independent of VE-cadherin gap formation.

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.