关键词： Cell culture  

摘要： During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca2+/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca2+, and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca2+ reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca2+, and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.

关键词： Phosphorylation   Endothelial cells   Immunoprecipitation   Small interfering RNA   Angiogenesis   Tyrosine   VEGF signaling   Protein kinases  

摘要： Background: Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood. Methodology/Principal Findings: In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM). Conclusions/Significance: Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2;it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regul

关键词： Ovarian Cancer   Ovarian Carcinoma   Ovarian Cancer Cell   Ovarian Cancer Cell Line   Cell Migration Assay  

摘要： Background: IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells. Methods: We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay. Results: IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAimediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration. Conclusion: Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.

关键词： IQ序列三磷酸鸟苷酶激活蛋白   细胞分裂周期42蛋白   气态甲醛   气道上皮   损伤修复  

摘要： 目的探讨包含IQ序列的三磷酸鸟苷酶(GTP)激活蛋白1(IQGAP1)和细胞分裂周期42蛋白(Cdc42)在气道上皮细胞损伤修复不同阶段的作用。方法选用健康昆明种小鼠24只,5 mg/m3气态甲醛,染毒4 h/d,分别染毒3,7和60 d后处死,采用免疫组织化学染色、蛋白印迹(Western blot)检测和免疫荧光共聚焦成像方法,观察IQ-GAP1和Cdc42蛋白在染毒小鼠气道上皮细胞损伤修复过程中的表达和定位变化。结果IQGAP1在染毒3和7 d时表达明显增强(P<0.01),60 d时恢复至对照组水平;Cdc42蛋白在染毒3 d时表达明显减弱(P<0.01),7 d时表达有所恢复。对照组中IQGAP1主要定位于气道上皮细胞的胞膜处,而Cdc42蛋白弥散分布于气道上皮细胞(AECs)胞质中;3 d和7 d时共同定位于细胞极化前缘;60 d时其定位与对照组相似。结论Cdc42蛋白和IQGAP1在气道上皮细胞损伤修复过程中的表达及定位呈动态变化,提示其在这一过程中发挥着重要作用。

关键词： Calcium ions   Cytoskeleton   EF-hand   IQ motif   Mlc1sa   Native gel  

摘要： IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.

关键词： actin cytoskeleton   cleaving embryo   meiotic spindle   oocyte   maturation   Cdc42   IQGAP1   toxin B   cytokinesis   oogenesis  

摘要： Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 Was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and Mitotic Cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte. (c) 2008 Elsevier Inc. All rights reserved.

关键词： scleroderma   lung fibrosis   connective tissue growth factor   cell migration  

摘要： Bogatkevich GS, Ludwicka-Bradley A, Singleton CB, Bethard JR, Silver RM. Proteomic analysis of CTGF- activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration. Am J Physiol Lung Cell Mol Physiol 295: L603-L611, 2008. First published August 1, 2008;doi: 10.1152/ajplung.00530.2007.-Connective tissue growth factor ( CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF, we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using 2-dimensional gel electrophoresis followed by in-gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF- induced proteins, such as pro-alpha ( I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF- responsive molecules that may have important roles in ILD. Our study demonstrates that 1 of the novel CTGF- induced proteins, IQ motif containing GTPase activating protein ( IQGAP) 1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF- induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF- induced cell migration and did not interfere with CTGF- induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further impli