关键词： Calcium-binding Proteins   Calmodulin   Cytoskeleton   MAPKs   Signal Transduction   Protein-Protein Interaction  

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。 (6)结构活性型RhoC和IQGAP1可以明显促进胃癌细胞vimentin和MMP-2的表达,抑制ICAM-1表达。 (7)干扰RhoC的表达

摘要： Leukocyte extravasation is a fundamental process of the inflammatory responses. The mechanisms that control remodelling of endothelial (EC) shape and adhesive contacts during leukocyte transendothelial migration (TEM) are not completely understood. We studied the role of EC phosphatidylinositol 3-kinase (PI3K) activity in lymphocyte TEM under shear stress conditions. Inhibition of EC PI3K activity by its pan inhibitors decreased lymphocyte diapedesis in a step after VE-cadherin opening. The importance of PI3K catalytic isoforms (p110α, p110β, p110δ and p110γ) were studied in TEM. Treatment of EC with isoform inhibitors of p110β, p110δ and p110γ did not affect lymphocyte TEM. Inhibition of p110α activity or expression reduced lymphocyte diapedesis. PI3K activity was measured in EC exposed to shear stress alone or shear stress on cells where ICAM-1 or VCAM-1 were cross-linked. The most significant effect was seen in cells cross-linked with ICAM-1 and exposed to shear stress. This suggests that cooperation of shear-induced mechanotransduction and ICAM-1 during leukocyte interaction with EC facilitates leukocyte diapedesis by inducing PI3K. We hypothesized that Rho GTP proteins downstream of PI3K activity are involved in leukocyte TEM. We studied the role of IQGAP1, a Rac1/Cdc42 effector, during lymphocyte TEM. EC IQGAP1 knockdown decreases both microtubule (MT) tethered to the adherens junction (AJ) and lymphocyte TEM. Similarly, loss of AJ-associated MT induced by brief nocodazole (ND) treatment decreases lymphocyte TEM. Neither intervention affected leukocyte migration to the interendothelial junctions. These data indicate that IQGAP1 contributes to MT stability at endothelial junctions and is involved in the junction remodelling required for efficient lymphocyte diapedesis. We studied a candidate Rho guanine nucleotide exchange factor named FGD5 upstream of IQGAP1. Inhibition of FGD5 expression resulted in more sensitivity to apoptotic stimuli and a higher rate of a

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);***

关键词： apoptosis   ECyd   focused proteomics   IQGAP1   RNase L  

摘要： Mammalian intracellular ribonuclease L ( RNase L) is a latent endoribonuclease that functions against viral infections as an apoptosis-inducing protein, and its activity requires intracellular 5'-end- triphosphorylated-2',5' oligoadenylates (2-5A) as an activator. Previously, we showed that RNase L can be activated in human cancer cell line HT1080 by an RNA polymerase I inhibitor, 1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl) cytosine (3'-ethynylcytidine;ECyd). In ECyd-treated cells, knockdown of the RNase L resulted in a marked decrease in c-jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting apoptosis. We investigate RNase L binding partners by focused proteomic approach using immunoprecipitation with anti-RNase L IgG and mass spectrometry. We found that the IQ motif-containing Ras GTPase-activating-like protein 1 (IQGAP1) can associate with RNase L, and that phosphorylation occurs on the IQGAP1. ECyd-induced JNK phosphorylation and apoptosis were inhibited when IQGAP1 was knocked down with a small interfering RNA. These results raise the interesting possibility that the RNase L-IQGAP1 association may regulate JNK phosphorylation in RNase L-madiated apoptosis. It is likely IQGAP1 works as a regulator in apoptosis.

关键词： Normal Liver   SNU387 Cell   Hepatic Adenoma   Liver Cancer Cell Line   Calponin Homology Domain  

摘要： Background: IQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC. Methods: IQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA. Results: IQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6). Conclusions: We conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.

关键词： Macrophages   Ischemia   Vasculogenesis   Inflammation   Angiogenesis   Blood flow   Necrosis   Muscle regeneration  

摘要： Background: Neovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF)-induced ROS production and migration of cultured endothelial cells (ECs);however, its role in post-ischemic neovascularization is unknown. Methodology/Principal Findings: Ischemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3(+) macrophages and CD31(+) capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and a-actin positive arterioles. Furthermore, IQGAP1(-/-) mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM)-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1(-/-) mice. In vitro, IQGAP1(-/-) BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1(-/-) mice. Conclusions/Significance: IQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation-and angiogenesis-dependent ischemic cardiovascular d