关键词： Beta-Catenin   Nuclear Translocation   Nuclear Transport   Signal Transduction   Wnt Pathway   Wnt Signaling   Dvl   IQGAP   Ran   Importin  

摘要： Background: The molecular mechanisms underlying the -catenin nuclear localization remain unclear. Results: IQGAP1 is a regulator of -catenin nuclear localization. Conclusion: Importin-5 and Ran contribute to the nuclear localization of -catenin. Significance: Novel molecular mechanisms were found in Wnt signaling. In the canonical Wnt signaling pathway, the translocation of -catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of -catenin function via importin-5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of -catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and -catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of -catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of -catenin in the canonical Wnt signaling pathway.

关键词： 血管紧张素Ⅱ   IQGAP1   肾小球损伤  

摘要： 目的研究血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)输注及替米沙坦干预对大鼠肾小球IQ domain GTPase-activating protein1(IQGAP1)表达的影响,并探讨IQGAP1在AngⅡ诱导肾小球损伤中的作用。方法 36只SPF级雄性Wistar大鼠随机分为生理盐水输注组、AngⅡ输注组和替米沙坦干预组,每周末测量大鼠血压及24h尿蛋白量,分别于14、28天处死大鼠取肾,光镜、电镜观察肾组织病理学及超微结构改变;免疫组织化学法、免疫荧光法及免疫印迹法检测IQGAP1在肾小球的表达及分布。结果 AngⅡ输注7天时大鼠出现显著的血压升高和尿蛋白量增加,且随输注时间的延长,血压及尿蛋白量进行性增高。替米沙坦干预可显著降低血压,并减少尿蛋白量。肾组织病理学显示AngⅡ输注组出现轻度系膜细胞增殖和系膜基质增加,且超微结构显示足细胞裂隙膜变窄,足突融合,替米沙坦干预可显著减轻上述病理改变。生理盐水输注组IQGAP1低水平表达并沿毛细血管袢呈线性分布,AngⅡ输注后IQGAP1表达量显著增加(P<0.05),而替米沙坦干预可显著抑制IQGAP1的表达上调(P<0.05);相关分析显示IQGAP1表达量与尿白蛋白量呈正相关(r=0.645,P<0.05)。结论 IQGAP1表达上调可能在AngⅡ诱导肾小球损伤中发挥重要作用。

关键词： 含IQ模序的GTP酶活化蛋白1   多发性骨髓瘤   MAP激酶信号通路   细胞增殖   细胞外信号调节激酶  

摘要： 目的:探讨含IQ模序的GTP酶活化蛋白1(IQ motif-containing GTPase-activating protein 1,IQGAP1)在骨髓瘤细胞中的过表达及其机制。方法:RT-PCR和Western blotting检测RPMI8226和U266细胞IQGAP1表达;使用不同的寡核苷酸序列构建沉默人IQGAP1的shRNA质粒(clone 1、clone 2、clone 3和clone 4)、转染RPMI8226细胞,RT-PCR和Western blotting检测其IQGAP1表达;Western blotting检测RPMI8226-shIQGAP1组(clone 1)、RPMI8226-shRNA阴性对照组和RPMI8226未转染组细胞p-ERK1/2、ERK1/2、Akt、p-AKT、STAT3和p-STAT3水平,免疫共沉淀确定IQGAP1和ERK的关系。结果:IQGAP1在RPMI8226和U266骨髓瘤细胞株中过表达,使用shRNA表达质粒沉默RPMI8226细胞IQGAP1后,IQGAP1表达减少,在有或无血管内皮生长因子(VEGF)/白细胞介素6(IL-6)作用下检测RPMI8226-shIQGAP1组(clone 1)细胞增殖明显下降。进一步检测3组细胞p-ERK1/2、ERK1/2、AKT、p-AKT、STAT3和p-STAT3蛋白水平,与RPMI8226-shRNA阴性对照组和RPMI8226未转染组相比,RPMI8226-shIQGAP1组ERK1/2磷酸化水平下降70.2%,免疫共沉淀法明确在RPMI8226细胞中IQGAP1和ERK存在相互作用。结论:IQGAP1在骨髓瘤细胞中的过表达可能与MAPK途径的ERK相互作用有关。

摘要： Recruitment of specific molecules to a specific membrane site is essential for communication between specialized membranous organelles. In the present study, we identified IQGAP1 as a novel GDP-bound-Rab27a-interacting protein. We found that IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Cdc42. We also found that IQGAP1 regulates the endocytosis of insulin secretory membranes. Silencing of IQGAP1 inhibits both endocytosis and the glucose-induced redistribution of endocytic machinery, including Rab27a and its binding protein coronin 3. These processes can also be inhibited by disruption of the trimeric complex with dominant negative IQGAP1 and Cdc42. These results indicate that activation of Cdc42 in response to the insulin secretagogue glucose recruits endocytic machinery to IQGAP1 at the cell periphery and regulates endocytosis at this membrane

关键词： N-WASP   Arp2   3 complex   Cdc42   Rac1   actin   Forster resonance energy transfer  

摘要： IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which then activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound Cdc42 or Rac1, which also bind IQGAP1. Here we report the use of purified proteins for in vitro binding and actin polymerization assays, and Forster (or fluorescence) resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions among IQGAP1, N-WASP, actin, and either Cdc42 or Rac1. In pyrene-actin assembly assays containing N-WASP and Arp2/3 complex, IQGAP1 plus either small G protein cooperatively stimulated actin filament nucleation by reducing the lag time before 50% maximum actin polymerization was reached. Similarly, Cdc42 and Rac1 modulated the binding of IQGAP1 to N-WASP in a dose-dependent manner, with Cdc42 enhancing the interaction and Rac1 reducing the interaction. These in vitro reconstitution results suggested that IQGAP1 interacts by similar, yet distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. The physiological relevance of these multi-protein interactions was substantiated by 3-color FRET microscopy of live MDCK cells expressing various combinations of fluorescent N-WASP, IQGAP1, Cdc42, Rac1, and actin. This study also establishes 3-color FRET microscopy as a powerful tool for studying dynamic intermolecular interactions in live cells. (c) 2013 Wiley Periodicals, Inc.

关键词： cell migration   IQGAP1   PIP2   PIPKI gamma   polybasic motif  

摘要： Phosphatidylinositol 4,5 bisphosphate (PIP2) is a key lipid messenger for regulation of cell migration. PIP2 modulates many effectors, but the specificity of PIP2 signalling can be defined by interactions of PIP2-generating enzymes with PIP2 effectors. Here, we show that type I gamma phosphatidylinositol 4-phosphate 5-kinase (PIPKI gamma) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKI gamma is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP2 effector that directly binds PIP2 through a polybasic motif and PIP2 binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKI gamma or PIP2 binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKI gamma and IQGAP1 in the control of cell migration.

关键词： IQGAP1   pancreatic cancer   tissue microarray   immunohistochemistry  

摘要： BACKGROUND:Pancreatic cancer is a highly aggressive malignant tumor with the lowest survival rate.A better understanding of the molecular mechanisms which contribute to pancreatic cancer occurrence and progression will aid in the development of new approaches to the early diagnosis,prevention,and treatment of this deadly *** scaffold protein IQGAP1 shows elevated levels in a variety of cancer ***,we investigated whether or not IQGAP1 is also overexpressed in pancreatic ***:IQGAP1 expression was examined in pancreatic cancer and normal tissues adjacent to cancerous tissues(adjacent tissues)by Western blotting and real-time RT-PCR as well as in paraffin sections of tissue microarray by *** correlations between IQGAP1 expression and various clinicopathological characteristics were ***:Western blotting and real-time RT-PCR revealed that the levels of IQGAP1 protein and mRNA expression in pancreatic cancer tissues were significantly increased compared with adjacent *** analysis on tissue microarray showed that IQGAP1 protein expression was significantly higher in pancreatic cancer(80.0%,48/60)compared with adjacent tissues(18.3%,11/60)(P<0.001).Moreover,overexpression of IQGAP1 was shown to be associated with the grades of tumor differentiation(P<0.05).CONCLUSION:The overexpression of IQGAP1 may play an important role in pancreatic cancer occurrence and progression,and IQGAP1 may serve as a novel molecular target for the diagnosis and treatment of pancreatic cancer.

关键词： fluorescence recovery after photobleaching (FRAP)   FRAP   hydroxyurea (HU)   ionizing radiation (IR)   IQ-domain GTPase-activating protein 1 (IQGAP1)   IQGAP1   neural-Wiskott–Aldrich syndrome protein (N-WASp)   Nuclear actin   Nuclear transport   Rac1   Ras-related C3 botulinum toxin substrate 1 (Rac1)   region of interest (ROI)   ultraviolet (UV)  

摘要： Abstract: Actin, a constituent of the cytoskeleton, is now recognized to function in the nucleus in gene transcription, chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data implicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Rac1 with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hydroxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization. [Copyright &y& Elsevier]

关键词： Nuclear actin   IQGAP1   Rac1   Nuclear transport   FRAP  

摘要： Actin, a constituent of the cytoskeleton, is now recognized to function in the nucleus in gene transcription, chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data implicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Racl with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hydroxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization. (C) 2013 Elsevier B.V. All rights reserved.

关键词： IQGAP1   Temporal lobe epilepsy   Hippocampus   Epileptogenesis   Reorganization  

摘要： Repeated seizures induce permanent alterations in the hippocampal circuits in experimental models with intractable temporal lobe epilepsy. Sprouting and synaptic reorganization induced by seizures has been well-studied in the mossy fiber pathway. However, studies investigating sprouting and synaptic reorganization beyond the mossy fiber pathway are limited. The present study examined the biochemical changes of CA1 pyramidal neurons undergoing morphological changes after excitotoxicity-induced hippocampal CA3 neuronal death. IQ-domain GTPase-activating proteins (IQGAP1), is an effector of Rac1 and Cdc42 and an actin-binding protein, was upregulated in CA1 pyramidal neurons after kainic acid-induced hippocampal CA3 neuronal degeneration. IQGAP1 + cells were colocalized with Nestin, but not in astrocytes or mature neurons. Furthermore, IQGAP1 did not originate from newly divided local precursors or NG2 + cells. IQGAP1 and adenomatous polyposis coli localized in CA1 pyramidal neurons, and Cdc42 activation was followed by IQGAP1 recruitment. These findings suggest that IQGAP1 is upregulated in pre-existed sparing neurons of the CA1 layer undergoing morphological changes after excitoxicity-induced hippocampal CA3 neuronal death. It demonstrates the utility of IQGAP1 as a possible marker for spared pyramidal neurons, which may contribute to structural and functional alternations responsible for the development of epilepsy.