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

关键词： Rho GTPases   IQGAP1   Proliferation   Injury and repair   Bronchial epithelial cells (BECs)  

摘要： The process of injury and repair involves spreading, migration and cell proliferation. The functions of Rho GTPases and their effector IQGAP1 are poor known in this process of airway epithelium. In the present study, we employed a widely used in vitro model by scratching a monolayer of BECs. We found that scratching induced decreasing of the GTP-bound Rac1 and Cdc42, but increasing the amounts of IQGAP1 at different time points. Next, we confirmed that IQGAP1 interacted with the constitutively active Rac1 (Rac1(V12)) and Cdc42(Cdc42(V12)) rather than the dominant negative Rac1 (Rac1(N17)) and Cdc42 (Cdc42(N17)). Over-expressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was defective to interact with Rho GTPases, induced translocation of beta-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and increased the expression levels of its target genes of c-myc and cyclin D1. Likewise, the amounts of c-myc and cyclin D1 increased after scratching. Our results suggested that IQGAP1 mediated cell proliferation through activating Tcf in a manner independent of Racl and Cdc42 in wound repair of BECs. (C) 2008 Elsevier Inc. All rights reserved.

关键词： CALMODULIN   EPIDERMAL growth factor   ORGANIC compounds   GROWTH factors   MUSCLE contraction   BIOLOGICAL transport  

摘要： Ca2+ and calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that Ca2+ and calmodulin regulate the MAPK signaling pathway at multiple positions in the cascade, but the molecular mechanism underlying these observations is poorly defined. We previously documented that IQGAP1 is a scaffold in the MAPK cascade. IQGAP1 binds to and regulates the activities of ERK, MEK, and B-Raf. Here we demonstrate that IQGAP1 integrates Ca2+ and calmodulin with B-Raf signaling. In vitro analysis reveals that Ca2+ promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by calmodulin in a Ca2+-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the Ca2+ ionophore A23187. In contrast, chelation of intracellular free Ca2+ concentrations ([Ca2+](i)) significantly enhances EGF-stimulated B-Raf activity, an effect that is dependent on IQGAP1. Incubation of cells with EGF augments the association of B-Raf with IQGAP1. Moreover, Ca2+ regulates the association of B-Raf with IQGAP1 in cells. Increasing [Ca2+](i) with Ca2+ ionophores significantly reduces co-immunoprecipitation of B- Raf and IQGAP1, whereas chelation of Ca2+ enhances the interaction. Consistent with these findings, increasing and decreasing [Ca2+](i) increase and decrease, respectively, co-immunoprecipitation of calmodulin with IQGAP1. Collectively, our data identify a previously unrecognized mechanism in which the scaffold protein IQGAP1 couples Ca2+ and calmodulin signaling to B-Raf function.

关键词： glioma   gene array   cadherin   catenin   IQGAP1   RhoA  

摘要： Abstract Transforming growth factor-α (TGF-α) and its receptor are frequently co-expressed in high-grade astrocytomas, suggesting a role for TGF-α autocrine/paracrine loops in the malignant progression of astrocytomas. To identify genes that may be critical in mediating TGF-α impact on the malignant progression of astrocytomas, we have used cDNA arrays to investigate TGF-α effects on the gene expression profile of U-373 MG glioblastoma cells. We found that in these cells ∼ 50 % of the TGF-α regulated genes code for cell motility/invasion-related proteins. TGF-α action on the expression of four of these proteins, α-catenin, IQGAP1, RhoA, and cadherin-11, was further investigated by immunoblotting in four astrocytoma cell lines and in normal astrocytes. The results demonstrate that the effects of TGF-α on IQGAP1, α-catenin, and RhoA expression are cell-line dependent. On the other hand, under TGF-α treatment, cadherin-11 expression is consistently decreased in all astrocytoma cell lines tested but is increased in normal astrocytes. In addition, we found that cadherin-11 is consistently down-regulated in astrocytomas versus normal brain tissues. Altogether, these results suggest that the down-regulation of cadherin-11 is a frequent molecular event in the neoplastic transformation of astrocytes and that this down-regulation may be initiated and/or amplified by TGF-α autocrine/paracrine loops during tumor progression.

关键词： distal convoluted tubule   collecting duct   macula densa   thick ascending limb   F-actin   cyclooxygenase 2   anion exchanger 1   calbindinD28k  

摘要： IQGAP1 is a multifunctional junction molecule that is involved in cell migration, proliferation, differentiation, cell polarity, and cell-cell adhesion. It is highly expressed in the kidney and has recently been identified in the glomerular basement membrane as a nephrin-associated protein. However, the distribution of IQGAP1 in renal tubular epithelial cells is unknown. We performed confocal microscopic studies to localize IQGAP1 in each nephron segment using dual immunofluorescence staining with various antibodies against segment-specific markers. We found that IQGAP1 was strongly expressed in the distal convoluted tubule (DCT), collecting duct, and macula densa and moderately in the thick ascending limb and proximal tubule. In the DCT, the IQGAP1-F-actin complex forms a comb-like structure with multiple parallel spikes sitting on the basal membrane. In the macula densa cells, IQGAP1 is strongly expressed in the apical membrane, whereas in type A intercalated cells, IQGAP1 is expressed in the basolateral membrane, where it colocalizes with anion exchanger 1, and in principal cells, it is diffusely expressed. In conclusion, we showed the expression and subcellular localization of IQGAP1 in various nephron segments. The site-specific expression pattern of this potent modulator of multiple biological pathways in the renal tubules suggests that IQGAP1 may have multiple important roles in various renal functions.

关键词： Neoplasms   degradation   Membranes   Cells   Actins  

摘要： Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.

关键词： IQGAP1   损伤修复   气道上皮细胞  

摘要： 气道上皮损伤修复过程包括细胞延伸、迁移和增殖。IQGAP1（IQ domain GTPase-activating protein1）是一个在许多细胞生命活动中非常有意义的蛋白，但其在肺上皮细胞中的作用尚未阐述清楚。本文采用目前广泛应用的刮伤气道上皮细胞的体外模型来研究IQGAP1的功能。结果显示，IQGAP1在小鼠、大鼠、猪和人气道上皮细胞中有丰富表达。它与微管骨架共定位，可被微管解聚剂nocodazole破坏。刮伤6-9h后，IQGAP1 mRNA及蛋白表达上调。过表达外源性IQGAP1导致β-catenin核转位，从而活化Tcf／Lef信号。此外，刮伤还影响IQGAP1与β-catenin、结肠腺瘤病（adenomatous polyposis coli，APC）蛋白及细胞质连接蛋白-170（cytoplasmic linker protein-170，CLIP-170）之间的相互作用。通过小于扰RNA（small interference RNA，siRNA）沉默IQGAP1表达则明显延迟损伤愈合。结果提示，IQGAP1信号参与气道上皮细胞损伤修复过程。

关键词： E-cadherin   gastric carcinoma   IQGAP1   Rac1   Rho GTPase   Tiam1  

摘要： Rho GTPases are a family of major regulators of E-cadherin-mediated cell adhesion that are implicated in the carcinogenic process by deregulated expression of the family members itself or of upstream modulators or downstream effectors. Combined investigation of the Rho GTPase Rac1, the effector protein IQGAP1 and the activator Tiam1 in relation to expression or mutation of E-cadherin in gastric adenocarcinomas has not been reported. The aim of the study was to determine the expression and prognostic significance of Rac1, IQGAP1, Tiam1 and E-cadherin in gastric adenocarcinomas. Gastric carcinomas of 76 patients were investigated immunohistochemically in a tissue microarray study for expression of Rac1, IQGAP1, Tiam1 and E-cadherin. Correlations with clinical and follow-up data were examined. Moderate or strong reactivity for Rac1 was observed in 46% and for Tiam1 in 56% of tumors. Expression of IQGAP1 was present in 59% and of E-cadherin in 87% of tumors. While Rac1 and E-cadherin expression were not related to prognosis, a trend was observed between a lack of IQGAP1 expression (log-rank 0.088) as well as presence of Tiam1 (log-rank 0.097) and favorable prognosis in Kaplan-Meier survival analysis. Expression of Rac1 was positively linked to IQGAP1 expression (P = 0.007, r = 0.343) and tended to be inversely associated with expression of E-cadherin (P = 0.055, r = -0.245). In conclusion, we observed deregulated expression of Rac1, IQGAP1, Tiam1 and E-cadherin in gastric cancer. We present evidence that either upregulation (for Rac1 and IQGAP1) or downregulation (for Tiam1 and E-cadherin) occurs. Rac1 and E-cadherin expression were not related to prognosis, while trends pointing to favorable prognosis of patients with Tiam1 expression and a lack of IQGAP1 expression were observed. These results indicate that the investigated regulators of E-cadherin-mediated cell adhesion play a role in gastric carcinogenesis.