关键词： endothelium   IQGAP1   Rac1   angiopoietin   permeability  

摘要： Objective-IQ domain GTPase-activating protein 1 (IQGAP1) contributes to cytoskeletal network regulation in epithelial cells by its scaffolding properties and by binding the Rho GTPase Rac1 to maintain its activity. The functions of IQGAP1 in endothelial cells beyond angiogenesis remain unclear. We hypothesized that IQGAP1 participates in the regulation of endothelial barrier function. Methods and Results-Silencing IQGAP1 in human microvascular endothelial cells resulted in a disruption of adherens junctions, formation of interendothelial gaps, and a reduction in barrier function. Furthermore, silencing of IQGAP1 abrogated the barrier enhancement effect of angiopoietin-1 (Angpt-1) and abolished the barrier-stabilizing effect of Angpt-1 on thrombin-stimulated cells. Coimmunoprecipitation detected binding of endogenous IQGAP1 with Rac1 at baseline that was stronger when Rac1 was activated and weaker when it was deactivated. Measurement of GTP-bound Rac1 revealed that Angpt-1 failed to activate Rac1 not only if IQGAP1 was silenced but also if cells were transfected with a mutant disabled in Rac1 binding (T1050AX2). Furthermore, a dominant-active Rac1 was sufficient to completely reverse the morphological and functional changes induced by reduction in IQGAP1. Conclusion-These experiments are the first demonstration of IQGAP1 regulating barrier function in any cell type. Further, our data show that Angpt-1 requires IQGAP1 as an indispensable activator of Rac1. (Arterioscler Thromb Vasc Biol. 2011;31:2643-2652.)

关键词： ERK1/2   FAK   IQGAP1   Melusin   Heart hypertrophy  

摘要： Extracellular signal-regulated kinase 1/2 (ERK1/2) signalling is a key pathway in cardiomyocyte hypertrophy and survival in response to many different stress stimuli. We have previously characterized melusin as a muscle-specific chaperone protein capable of ERK1/2 signalling activation in the heart. Here, we show that in the heart, melusin forms a supramolecular complex with the proto-oncogene c-Raf, MEK1/2 (also known as MAPKK1/2) and ERK1/2 and that melusin-bound mitogen-activated protein kinases (MAPKs) are activated by pressure overload. Moreover, we demonstrate that both focal adhesion kinase (FAK) and IQ motif-containing GTPase activating protein 1 (IQGAP1), a scaffold protein for the ERK1/2 signalling cascade, are part of the melusin complex and are required for ERK1/2 activation in response to pressure overload. Finally, analysis of isolated neonatal cardiomyocytes indicates that both FAK and IQGAP1 regulate melusin-dependent cardiomyocyte hypertrophy and survival through ERK1/2 activation.

关键词： Actin   Calmodulin   Cdc42   Cell Motility   Signal Transduction   Barbed End Capping   IQGAP1   N-WASP  

摘要： IQGAP1 is a large modular protein that displays multiple partnership and is thought to act as a scaffold in coupling cell signaling to the actin and microtubule cytoskeletons in cell migration, adhesion, and cytokinesis. However the molecular mechanisms underlying the activities of IQGAP1 are poorly understood in part because of its large size, poor solubility and lack of functional assays to challenge biochemical properties in various contexts. We have purified bacterially expressed recombinant human IQGAP1. The protein binds Cdc42, Rac1, and the CRIB domain of N-WASP in a calmodulin-sensitive fashion. We further show that in addition to bundling of filaments via a single N-terminal calponin-homology domain, IQGAP1 actually regulates actin assembly. It caps barbed ends, with a higher affinity for ADP-bound terminal subunits (K-B = 4 nM). The barbed end capping activity is inhibited by calmodulin, consistent with calmodulin binding to IQGAP1 with a K-C of 40 nM, both in the absence and presence of Ca2+ ions. The barbed end capping activity resides in the C-terminal half of IQGAP1. It is possible that the capping activity of IQGAP1 accounts for its stimulation of cell migration. We further find that bacterially expressed recombinant IQGAP1 fragments easily co-purify with nucleic acids that turn out to activate N-WASP protein to branch filaments with Arp2/3 complex. The present results open perspectives for tackling the function of IQGAP1 in more complex reconstituted systems.

关键词： IQGAP1   肝癌细胞   mTOR  

摘要： 目的:研究IQGAP1(IQ domain GTPase-activating proteins,IQGAP1)对肝癌细胞株增殖及mTOR信号转导通路的影响,阐述其诱导细胞增殖的机制。方法:Western blot检测IQGAP1在肝癌细胞株HepG2、Huh-7中的表达。选择高表达IQGAP1的细胞株,针对IQGAP1基因的mRNA序列合成siRNA,采用脂质体转染法将其转入HepG2细胞;采用MTT法分析细胞的增殖;流式细胞仪检测细胞周期变化;免疫印迹法观测IQGAP1、mTOR和p-mTOR蛋白表达的变化。结果:IQGAP1在HepG2和Huh-7细胞中均表达,且在HepG2细胞中表达高于Huh-7细胞。IQGAP1 siRNA转染HepG2细胞后,抑制细胞的增殖;DNA合成前期(G_0/G_1期)细胞比例上升,合成期(S期)细胞比例下降;mTOR表达变化不明显,而p-mTOR的表达下降。结论:IQGAP1促进肝癌细胞的增殖,其机制可能是通过mTOR相关信号通路实现的。

关键词： Thymosin beta(4) (T beta(4))   ILK   IQGAP1   Rac1   Migration  

摘要： Thymosin beta(4) (T beta(4)) overexpression increases cell migration and tumor metastasis. Hence, understanding the mechanism of cancer cell migration induced by T beta(4) may provide means to inhibit their metastasis. We demonstrated higher Rac1 activities and expression levels of IQGAP1 and ILK in highly migrated T beta(4)-overexpressing SW480 cells. In addition, IQGAP1 formed a complex with ILK and knockdown of T beta(4) simultaneously reduced ILK and IQGAP1 protein levels as well as their migration ability. These findings suggest that T beta(4) increases migration of colon cancer cells via activating Rac1 by elevating IQGAP1/ILK complexes and IHC results illustrated a similar mechanism occurring in vivo. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

关键词： Immune synapse   IQGAP1   MTOC   NK cells   shRNA  

摘要： Natural killer (NK) cells form a region of tight contact called the NK immunological synapse (NKIS) with their target cells. This is a dynamic region serving as a platform for targeted signaling and exocytotic events. We previously identified IQGAP1 as a cytoskeletal component of the NK-like cell line YTS. The present study was undertaken to determine the role of IQGAP1 in the function of NK cells. Silencing of IQGAP1 expression resulted in almost complete loss of the cytotoxic activity of YTS cells. Loss of IQGAP1 did not prevent conjugate formation with target cells but it did result in a failure to reorient the microtubule organizing centre to the immune synapse. Significantly, IQGAP1 expression was required for the perigranular accumulation of an F-actin network. IQGAP1 was shown to undergo marked rearrangements during synapse maturation in effector target conjugates of YTS or primary NK cells. These results suggest previously undescribed role(s) for IQGAP1 in regulating multiple aspects of cytoskeletal organization and granule polarization in NK cells.

关键词： Breast Cancer   Calmodulin   Cancer Therapy   Signal Transduction   Tumor Marker   IQGAP1   Trastuzumab  

摘要： Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast cancers. Increased HER2 expression is an adverse prognostic factor and correlates with decreased patient survival. HER2-positive (HER2(+)) breast cancer is treated with trastuzumab. Unfortunately, some patients are intrinsically refractory to therapy, and many who do respond initially become resistant within 1 year. Understanding the molecular mechanisms underlying HER2 signaling and trastuzumab resistance is essential to reduce breast cancer mortality. IQGAP1 is a ubiquitously expressed scaffold protein that contains multiple protein interaction domains. By regulating its binding partners IQGAP1 integrates signaling pathways, several of which contribute to breast tumorigenesis. We show here that IQGAP1 is overexpressed in HER2(+) breast cancer tissue and binds directly to HER2. Knockdown of IQGAP1 decreases HER2 expression, phosphorylation, signaling, and HER2-stimulated cell proliferation, effects that are all reversed by reconstituting cells with IQGAP1. Reducing IQGAP1 up-regulates p27, and blocking this increase attenuates the growth inhibitory effects of IQGAP1 knockdown. Importantly, IQGAP1 is overexpressed in trastuzumab-resistant breast epithelial cells, and reducing IQGAP1 both augments the inhibitory effects of trastuzumab and restores trastuzumab sensitivity to trastuzumab-resistant SkBR3 cells. These data suggest that inhibiting IQGAP1 function may represent a rational strategy for treating HER2(+) breast carcinoma.