关键词： EPIDERMAL growth factor   PROTEIN-tyrosine kinase   CALMODULIN   PHOSPHORYLATION   MASS spectrometry  

摘要： Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca2+ attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKC alpha isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.

关键词： miR-124a   GBM   Migration   Invasion   Overall survival   IQGAP1   LAMC1   ITGB1  

摘要： Glioblastoma (GBM) represents a formidable clinical challenge for both patients and treating physicians. Due to better local treatments and prolonged patient survival, remote recurrences are increasingly observed, underpinning the importance of targeting tumour migration and attachment. Aberrant expression of microRNA (miRNA) is commonly associated with cancer and loss of miR-124a has previously been implicated to function as a tumour suppressor. The assessment of miR-124a in clinical specimens has been limited and a potential role in migration and invasion has been unexplored until now. We measured the expression levels of mature miR-124a in a retrospective series of 119 cases of histologically confirmed GBM and found its expression was markedly lower in over 80% of the GBM clinical specimens compared to normal brain tissue. The level of reduction in the clinical cohort varied significantly and patients with lower than the average miR-124a expression levels displayed shorter survival times. Endogenous miR-124a expression and the protein expression of three of its targets;IQ motif containing GTPase activating protein 1 (IQGAP1), laminin gamma 1 (LAMC1) and integrin beta 1 (ITGB1) were significantly reciprocally associated in the majority of the clinical cases. We confirmed this association in our in vitro model. Functionally, the ectopic expression of mature miR-124a in a GBM cell line resulted in significant inhibition of migration and invasion, demonstrating a role for miR-124a in promoting tumour invasiveness. Our results suggest that miR-124a may play a role in GBM migration, and that targeted delivery of miR-124a may be a novel inhibitor of GBM invasion. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

关键词： Virulence   Pathogenesis   Attenuation   Core protein   Classical swine fever virus  

摘要： Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J. 2006 25:2155]. Alanine-substitution within Core regions I, II. III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues (207)AT(209) (I), (VVE212)-V-210 (II), (213)GVK(215) (III), or (232)GLYHN(236) (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle. Published by Elsevier Inc.

关键词： SYNAPSES   MICROTUBULES   NEURONS   ACTIN   CYTOSKELETON  

摘要： Dendritic arbors are compartments of neurons dedicated to receiving synaptic inputs. Their shape is an outcome of both the intrinsic genetic program and environmental signals. The microtubules and actin cytoskeleton are both crucial for proper dendritic morphology, but how they interact is unclear. The present study demonstrates that microtubule plus-end tracking protein CLIP-170 and actin-binding protein IQGAP1 regulate dendrite morphology of rat neurons by coordinating the interaction between microtubules and the actin cytoskeleton. Moreover, we show that mTOR kinase interacts with CLIP-170 and is needed for efficient formation of a protein complex containing CLIP-170 and IQGAP1. Dynamic microtubules, CLIP-170, and IQGAP1 are required for proper dendritic arbor morphology and PI3K-mTOR-induced increase in dendritic arbor complexity. Moreover, CLIP-170 and IQGAP1 knockdown modulates dendritic arbor growth via regulation of the actin cytoskeleton. We postulate that mTOR controls dendritic arbor morphology by enhancing cross talk between dynamic microtubules and actin through CLIP-170 and IQGAP1.

关键词： EPEC   IQGAP1   Microbial pathogenesis   Salmonella  

摘要： Microbial pathogens cause widespread morbidity and mortality. Central to the pathogens' virulence is manipulation of the host cell's cytoskeleton, which facilitates microbial invasion, multiplication, and avoidance of the innate immune response. IQGAP1 is a ubiquitously expressed scaffold protein that integrates diverse signaling cascades. Research has shown that IQGAP1 binds to and modulates the activity of multiple proteins that participate in bacterial invasion. Here, we review data that support a role for IQGAP1 in infectious disease via its ability to regulate the actin cytoskeleton. In addition, we explore other mechanisms by which IQGAP1 may be exploited by microbial pathogens. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

关键词： CARRIER proteins   INTRACELLULAR pathogens   CYTOSKELETON   CELL membranes   TYROSINE   PROTEINS  

摘要： Ca2+-binding proteins of the S100 family participate in intracellular Ca2+ signaling by binding to and regulating specific cellular targets in their Ca2+-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca2+-dependent and characterized by a dissociation constant of 0.2 mu M. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca2+-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.

关键词： 肺癌   IQGAP1   细胞运动   侵袭转移  

摘要： 目的探讨肺癌中支架蛋白IQGAP1的蛋白表达水平,及IQGAP1对肺癌细胞运动侵袭能力的影响。方法 RT-PCR检测IQGAP1mRNA的表达,免疫组织化学SABC法及Western blot检测IQ-GAP1蛋白的表达,并采用Transwell实验探讨IQGAP1蛋白促进侵袭转移的作用机制。结果研究发现其在肺癌组织存在高表达,IQGAP1蛋白可以通过提高肺癌细胞的运动能力促进肺癌的侵袭转移。结论 IQGAP1与肺癌密切相关,可能在肺癌的侵袭和转移中起重要做用。

关键词： IQGAP1   Nuclear transport   beta-Catenin   Cell cycle   S phase  

摘要： IQGAP1 is a plasma membrane-associated protein and an important regulator of the actin cytoskeleton, contributing to cell migration, polarity and adhesion. In this study, we demonstrate the nuclear translocation of IQGAP1 using confocal microscopy and cell fractionation. Moreover, we identify a specific pool of IQGAP1 that accumulates in the nucleus during late G1-early S phase of the cell cycle. The nuclear targeting of IQGAP1 was facilitated by N- and C-terminal sequences, and its ability to slowly shuttle between nucleus and cytoplasm/membrane was partly regulated by the CRM1 export receptor. The inhibition of GSK-3 beta also stimulated nuclear localization of IQGAP1. The dramatic nuclear accumulation of IQGAP1 observed when cells were arrested in G1/S phase suggested a possible role in cell cycle regulation. In support of this, we used immunoprecipitation assays to show that the nuclear pool of IQGAP1 in G1/S-arrested cells associates with DNA replication complex factors RPA32 and PCNA. More important, the siRNA-mediated silencing of IQGAP1 significantly delayed cell cycle progression through S phase and G2/M in NIH 3T3 cells released from thymidine block. Our findings reveal an unexpected regulatory pathway for IQGAP1, and show that a pool of this cytoskeletal regulator translocates into the nucleus in late G1/early S phase to stimulate DNA replication and progression of the cell cycle. (C) 2010 Elsevier Ltd. All rights reserved.

关键词： RhoC   IQGAP1   胃癌   迁移   增殖  

摘要： 研究目的 (1)检测RhoC和IQGAP1 (IQ-domain GTPase-activating protein1)在胃癌组织和胃癌细胞株中的表达,分析RhoC和IQGAP1的相关性以及两者与临床病理参数的相关性。 (2)研究RhoC和IQGAP1在调控胃癌细胞迁移、增殖过程中的作用以及在调控过程中两者之间的相互关系。 研究方法 (1)分别将构建的编码IQGAP1和IQGAP1-C基因的腺病毒载体以及获赠的编码结构活性型RhoC基因的腺病毒载体转染293A细胞,制备病毒,并对病毒滴定以及感染后目的蛋白的表达进行检测,选择对重组腺病毒敏感的胃癌细胞株进行后续实验。 (2)采用RT-PCR、Western blotting、免疫荧光等方法,检测IQGAP1和RhoC在胃癌组织和细胞株中的表达；利用Spearman's rank相关性分析分析胃癌组织中RhoC与IQGAP1表达的相关性；利用Fisher确切概率法,分析两者的表达与临床病理参数相关性。 (3)通过感染腺病毒pAd-RhoC-V14,增加细胞内结构活性型RhoC的表达,采用MTT实验分析RhoC对胃癌细胞增殖的影响；采用transwell迁移实验分析RhoC对胃癌细胞迁移的影响。 (4)将质粒Flag-IQGAP1、Flag-IQGAP1-C或Flag-IQGAP1-N(分别编码IQGAP1全长、C末端或N末端)与绿色荧光蛋白(GFP)质粒共同转染胃癌细胞BGC-823,采用BrdU掺入实验分析不同结构的IQGAP1对单细胞增殖的影响。 (5)通过感染编码IQGAP1(编码IQGAP1全长)或IQGAP1-C(编码IQGAP1的C末端,氨基酸：860-1657)基因的腺病毒,增加胃癌细胞内IQGAP1或IQGAP1-C的表达,采用MTT实验分析IQGAP1或IQGAP1-C在胃癌细胞增殖中的作用；采用trans well迁移实验分析IQGAP1或IQGAP1-C在胃癌细胞迁移过程中的作用。 (6)通过转染RhoC-siRNA和IQGAP1-siRNA,分别干扰胃癌细胞中RhoC和IQGAP1蛋白的表达；采用MTT实验和transwell迁移实验,分别观察抑制RhoC或IQGAP1表达对胃癌细胞增殖和迁移的影响。 (7)采用Western blotting对细胞粘附、迁移相关蛋白ICAM-1、vimentin、MMP-2等进行检测,进而分析RhoC和IQGAP1对上述蛋白表达的影响。 (8)利用RhoC-siRNA干扰胃癌细胞内RhoC的表达,然后再将编码IQGAP1和IQGAP1-C基因的腺病毒感染胃癌细胞,通过transwell迁移实验和MTT实验分析干扰RhoC的表达是否影响IQGAP1刺激的胃癌细胞迁移和增殖；利用IQGAP1-siRNA干扰胃癌细胞内IQGAP1的表达,再将编码结构活性型RhoC基因的腺病毒感染胃癌细胞,通过transwell迁移实验和MTT实验分析干扰IQGAP1的表达是否影响RhoC刺激的胃癌细胞迁移和增殖。 (9)分别将腺病毒pAd-IQGAP1或者pAd-IQGAP1-C与pAd-RhoC-V14共同感染非洲绿猴SV40转化的肾细胞COS-7和胃癌细胞BGC-823,通过免疫荧光和免疫共沉淀方法,观察RhoC与IQGAP1在细胞中的定位和结合情况。 研究结果 (1)在29例胃癌组织标本中,RhoC和IQGAP1的表达高于对应癌旁组织者有22例,占总例数的75.9%(22/29)；IQGAP1和RhoC在胃癌组织中的表达水平均与肿瘤分化程度负相关(P<0.05,P<0.05),与胃癌细胞的迁移、增殖、浸润有一定的相关性,但是差异没有统计学意义。 (2)利用制备的重组腺病毒pAd-IQGAP1、pAd-IQGAP1-C和pAd-RhoC-V14感染胃癌细胞；Western blotting结果显示,在感染上述腺病毒的胃癌细胞中IQGAP1、IQGAP1-C和RhoC蛋白的表达明显增加；在后续试验中,选取增殖能力较强的BGC-823细胞和迁移能力较强的AGS细胞分别做增殖和迁移分析。 (3)RhoC和IQGAP1在三种胃癌细胞株中的表达明显高于胃粘膜上皮细胞株GES-1；IQGAP1在胃癌细胞内的分布以在细胞膜和细胞与细胞粘连处居多。 (4)结构活性型RhoC能明显促进胃癌细胞的迁移和增殖,干扰RhoC的表达抑制了胃癌细胞迁移和增殖。 (5)IQGAP1和IQGAP1-C明显增强胃癌细胞迁移和增殖能力,而IQGAP1-N对胃癌细胞增殖的影响不明显。IQGAP1-siRNA可以显著抑制细胞迁移和增殖。

关键词： 肺炎衣原体   基因芯片   血管平滑肌细胞   细胞粘附   细胞迁移   IQGAP1shRNA  

摘要： 目的： 利用肺炎衣原体(Chlamdia pneumoniae, ***)体外感染大鼠血管平滑肌细胞(Vascular smooth muscle cell, VSMC)模型,分析***感染对VSMC(?)细胞迁移相关基因表达的影响：观察***感染对VSMC粘附和迁移能力的影响;探讨IQGAP1在***感染诱导VSMC粘附和迁移中的作用。 方法： ***增殖培养后体外感染大鼠VSMC;2.应用基因芯片技术分析***感染大鼠VSMC12h后,VSMC内细胞迁移相关基因表达的改变;3.运用MTT去观察***感染对VSMC粘附能力的影响;4.应用Transwell实验观察***感染对VSMC迁移能力的影响;5.利用RT-PCR技术检测***感染的VSMC内IQGAP1mRNA的表达水平;6.采用Western blot技术检测***感染的VSMC为IQGAP1蛋白的表达水平;7.应用RNA干扰技术使IQGAP1表达水平下调,RT-PCR技术检测IQGAP1mRNA的表达,以确定干扰效果;8.运用MTT法观察RNA干扰下调IQGAP1基因后对***感染诱导的VSMC粘附的影响;9.应用Transwell实验观察RNA干扰沉默IQGAP1表达后对***感染诱导的VSMC迁移的影响。 结果： ***感染组与正常对照组均应用Affymetrix Rat Genome2302.0Array,通过与大鼠31000个探针进行杂交,分析了31042个转录体和变异体,其中包括28000个己知基因。芯片杂交结果通过计算机扫描、定量和分析后,共发现差异表达基因35个,其中上调基因12个,下调基因23个;与细胞迁移密切相关的TLR2基因上调、TGF-B3和AKAP12基因下调。基因本体论分析结果显示,35个差异显著的基因中,30个基因可以在生物学过程、细胞组成和分子功能中找到注释,4个基因未搜索到其功能分类;代谢通路分析结果显示,7个基因参与细胞周期相关的信号通路、补体激活的经典途径相关信号通路、氧化应激相关信号通路和前列腺素合成调节相关信号通路;***感染VSMC16h后,细胞粘附于Matrigel的能力明显增强。MTT比色结果显示,***感染组的吸光度(Absorbance, A)值明显高于正常对照组(P<0.001);Cpn感染组的细胞粘附率为162.342%±4.691%(P<0.001);***感染VSMC19h后,***感染组的细胞迁移数目明显多于正常对照组(P<0.01);细胞迁移指数为152.653%±7.493%(P<0.01);***感染VSMC2h后,IQGAP1(?)带亮度与正常对照组差别不大;随着感染时间延长,IQGAP1条带亮度逐渐增强;***感染12h时IQGAP1条带亮度最强;之后,IQGAP1条带亮度又逐渐减弱,直至感染48h时,条带亮度与正常对照组基本相同;5. Western blot结果显示,***感染VSMC后,随着感染时间延长,IQGAP1的表达水平也整体表现出先逐渐升高而后又逐渐降低的趋势;***感染VSMC16h时IQGAP1的表达水平最高;随后,IQGAP1的表达水平又逐渐降低;6.重组质粒转染VSMC后24h,荧光显微镜下可观察到pGPH1/GFP/Neo-IQGAP1Scrambled shRNA转染组(阴性对照组)和pGPH1/GFP/Neo-IQGAP1shRNAs转染组细胞均发出清晰的绿色荧光,而无质粒转染对照组和正常对照组细胞中则未见有绿色荧光;转染48h后,各组细胞中的绿色荧光较24h时增强;RT-PCR结果显示,无质粒转染对照组和阴性对照组细胞内IQGAP1mRNA表达水平与正常对照组无显著差异;而IQGAP1mRNA在pGPH1/GFP/Neo-IQGAP1shRNA转染组中的表达水平与正常对照组相比明显降低： 7.经MTT比色后发现,pGPH1/GFP/Neo-IQGAP1shRNAs转染组的A值明显低于对照组(P<0.001),细胞粘附率为68.543%±1.197%(P<0.001);阴性对照组细胞粘附于Matrigel的能力与正常对照组相比无明显变化(P>0.05);***感染转染pGPH1/GFP/Neo-IQGAP1shRNAs的VSMC后,该组的A值明显低于***感染组(P<0.001),其细胞粘附率为104.991%±1.225%(P<0.001);8.重组质粒pGPHl/GFP/Neo-IQGAP1shRNAs转染VSMC后,细胞迁移数目明显低于正常对照组(P<0.01),细胞迁移指数为69.927%±4.838%(P<0.01);阴性对照组细胞的迁移数目与正常对照组相比无明显差异(P>0.05);***