摘要： Lis1 gene defects impair neuronal migration, causing the severe human brain malformation lissencephaly. Although much is known about its interactions with microtubules, microtubule-binding proteins such as CLIP-170, and with the dynein motor complex, the response of Lis1 to neuronal motility signals has not been elucidated. Lis1 deficiency is associated with deregulation of the Rho-family GTPases Cdc42, Rac1 and RhoA, and ensuing actin cytoskeletal defects, but the link between Lis1 and Rho GTPases remains unclear. We report here that calcium influx enhances neuronal motility through Lis1-dependent regulation of Rho GTPases. Lis1 promotes Cdc42 activation through interaction with the calcium sensitive GTPase scaffolding protein IQGAP1, maintaining the perimembrane localization of IQGAP1 and CLIP170 and thereby tethering microtubule ends to the cortical actin cytoskeleton. Lis1 thus is a key component of neuronal motility signal transduction that regulates the cytoskeleton by complexing with IQGAP1, active Cdc42 and CLIP-170 upon calcium influx.

关键词： 肝细胞癌   IQGAP1   细胞运动   侵袭转移  

摘要： 目的：研究肝细胞癌（HCC）组织中IQGAP1的表达情况与HCC临床病理特征以及随访预后的关系，探讨IQGAP1在HCC侵袭转移中的作用机制。 材料与方法：收集36例新鲜HCC标本以及112例HCC石蜡标本，所有标本按取材部位分为HCC组织和癌旁肝组织。将HCC组织标本均切成5微米厚的切片，HE染色观察肿瘤镜下微血管侵犯、肿瘤包膜完整性、肝硬化程度、肿瘤细胞分化程度等临床病理特征。按肿瘤结节数目及肿瘤直径将HCC标本分为三组：孤立性大肝癌组（指单个结节且肿瘤直径＞5厘米，SLHCC），小肝癌组（指单个结节或两个结节，肿瘤直径之和≤5cm，SHCC），结节性肝癌组（指肿瘤结节≥2个，两个结节且直径之和≤5厘米者除外，NHCC）。采用半定量逆转录多聚酶链式反应（RT-PCR）检测IQGAP1 mRNA的表达，免疫组织化学SABC法及Western blot检测IQGAP1蛋白的表达。根据IQGAP1在HCC细胞中表达的部位，将IQGAP1免疫组化阳性染色的HCC标本分为IQGAP1胞膜表达型和IQGAP1胞浆表达型。比较IQGAP1在各组HCC中的表达水平、表达类型以及与HCC临床病理特征关系，分析IQGAP1表达与HCC手术预后的关系。采用具有不同侵袭转移能力的HepG2细胞株和CCL13细胞株检测IQGAP1蛋白表达的差异。通过IQGAP1小鼠抗人单克隆抗体阻断HepG2细胞株中IQGAP1蛋白的功能，并采用Transwell实验探讨IQGAP1蛋白促进HCC侵袭转移的作用机制。 结果：显微镜下观察HE染色的病理切片，结果显示81.1％

关键词： actin cytoskeleton   endothelial cell migration   IQGAP1   NAD(P)H oxidase   reactive oxygen species  

摘要： Objective-Endothelial cell (EC) migration is a key event for repair process after vascular injury and angiogenesis. EC migration is regulated by reorganization of the actin cytoskeleton at the leading edge and localized production of reactive oxygen species (ROS) at the site of injury. However, underlying mechanisms are unclear. We reported that IQGAP1, an actin binding scaffold protein, mediates VEGF-induced activation of gp91phox (Nox2)-dependent NAD(P) H oxidase and EC migration. We thus hypothesized that Nox2 and IQGAP1 may play important roles in ROS-dependent EC migration in response to injury. Methods and Results-Using a monolayer scratch assay with confluent ECs, we show that ROS production is increased at the margin of scratch area and Nox2 translocates to the leading edge, where it colocalizes and associates with both actin and IQGAP1 in migrating ECs. Knockdown of IQGAP1 using siRNA and inhibition of the actin cytoskeleton blocked scratch injury-induced H2O2 production, Nox2 translocation and its interaction with actin, and EC migration toward the injured site. Conclusions-These suggest that IQGAP1 may function to link Nox2 to actin at the leading edge, thereby facilitating ROS production at the site of injury, which may contribute to EC migration.

关键词： Proteins  

摘要： The scaffolding protein IQGAP1 participates in numerous cellular functions by binding to target proteins such as actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. IQGAP1 regulates the cytoskeleton, promotes cell motility, and modulates E-cadherin-mediated cell-cell adhesion. However, how IQGAP1 exerts its functions in vivo is still unclear. In this study we investigate the self-association of IQGAP1 and its role in IQGAP1 function. Endogenous IQGAP1 co-immunoprecipitated from MCF-7 cells with IQGAP1 tagged with enhanced green fluorescent protein, indicating that IQGAP1 self-associates in cells. In vitro assays confirmed that IQGAP1 can self-associate and that this effect is mediated by the N-terminal half of the protein. Gel filtration analysis suggested that full-length IQGAP1 exists as a combination of monomers, dimers, and larger oligomers. Analysis performed with multiple fragments of IQGAP1 narrowed the self-association region to amino acids 763 - 863. In support of this observation, a peptide comprising residues 763 - 863 disrupted self-association of full-length IQGAP1 in a dose-dependent manner. Similarly, deleting this sequence from IQGAP1 abolished binding to full-length IQGAP1. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. Consistent with these findings, transfection into cells of a peptide containing the self-association domain significantly reduced the amount of active Cdc42 in cell lysates. These observations define a sequence of IQGAP1 that is necessary for its oligomerization and demonstrate that self-association is required for the normal cellular function of IQGAP1.

关键词： glutamate   interaction   phosphorylation   receptor   synapses   trafficking  

摘要： AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors play key roles in excitatory synaptic transmission and synaptic plasticity in the CNS. Although a variety of proteins has been characterized to interact with AMPA receptors and regulate their function, little is known about the regulation of the AMPA receptor subunit GluR4. To understand the molecular mechanisms of GluR4 functional regulation, the yeast two-hybrid system was used to identify GluR4-interacting molecules. alpha-Actinin-1 and IQGAP1 were identified to be GluR4-specific binding partners. Both proteins interact specifically with GluR4 and co-cluster with GluR4 individually in neurons. Mapping experiments revealed that alpha-Actinin-1 and IQGAP1 bind to the same region within the C-terminus of GluR4 that contains a previously identified PKA phosphorylation site, Ser842, phosphorylation of which is regulated by synaptic activity. Interestingly, the phosphorylation of Ser842 differentially regulates interactions of GluR4 with alpha-Actinin-1 and IQGAP1;phosphorylation strongly inhibits interaction of GluR4 with alpha-Actinin-1 but has little effect on its interaction with IQGAP1. These results suggest that alpha-Actinin-1 and IQGAP1 regulate GluR4 functions via their specific associations with GluR4. In addition, our data indicate that activity-dependent phosphorylation of GluR4 may regulate its synaptic targeting through phosphorylation-dependent interactions with alpha-Actinin-1 and IQGAP1.