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： NUCLEOTIDE EXCHANGE FACTOR   CELL-ADHESION   CDC42   CALMODULIN   PROTEIN   DIMERIZATION   BINDING   CYTOSKELETON   IDENTIFICATION   MOTIFS  

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.

关键词： CELL-CELL ADHESION   IN-VIVO   BINDING PARTNER   CROSS-LINKING   RAF-1 KINASE   CDC42   ACTIN   CALMODULIN   RAS   IDENTIFICATION  

摘要： IQGAP1 modulates many cellular functions such as cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds extracellular signal-regulated kinase (ERK) 2 and regulates growth factor-stimulated ERK activity. Here we show that MEK, the molecule immediately upstream of ERK in the Ras/mitogen-activated protein (MAP) kinase signaling cascade, also interacts directly with IQGAP1. Both MEK1 and MEK2 bound IQGAP1 in vitro and coimmunoprecipitated with IQGAP1. The addition of ERK2 enhanced by fourfold the in vitro interaction of MEK2 with IQGAP1 without altering binding of MEK1. Similarly, ERK1 promoted MEK binding to IQGAP1, but neither MEK protein altered the association between IQGAP1 and ERK. Epidermal growth factor (EGF) differentially regulated binding, enhancing MEK1 interaction while reducing MEK2 binding to IQGAP1. In addition, both knockdown and overexpression of IQGAP1 reduced EGF-stimulated activation of MEK and ERK. Analyses with selective IQGAP1 mutant constructs indicated that MEK binding is crucial for IQGAP1 to modulate EGF activation of ERK. Our data strongly suggest that IQGAP1 functions as a molecular scaffold in the Ras/MAP kinase pathway.

关键词： cell adhesion molecules   cell-cell junctions   glomerular epithelial cell   podocyte   slit diaphragm  

摘要： Nephrin is a cell surface receptor of the Ig superfamily that localizes to slit diaphragms, the specialized junctions between the interdigitating foot processes of the glomerular epithelium (podocytes) in the kidney. Mutations in the NPHS1 gene encoding nephrin lead to proteinuria and congenital nephrotic syndrome, indicating that nephrin is essential for normal glomerular development and function. To identify nephrin-binding proteins, we performed mass spectrometry on proteins obtained from pull-down assays with GST-nephrin cytoplasmic domain. Nephrin specifically pulled down six proteins from glomerular lysates, MAGI-2/S-SCAM (membrane-associated guanylate kinase inverted 2/synaptic scaffolding molecule), IQGAP1 (IQ motif-containing GTPase-activating protein 1), CASK (calcium/calmodulin-dependent serine protein kinase), a-actinin, all spectrin, and beta II spectrin. All of these scaffolding proteins are often associated with cell junctions. By immunofluorescence these proteins are expressed in glomerular epithelial cells, where they colocalize with nephrin in the foot processes. During glomerular development, IQGAP1 is expressed in the junctional complexes between the earliest identifiable podocytes, MAGI-2/S-SCAM is first detected in junctional complexes in podocytes after their migration to the base of the cells. Thus, the nephrin-slit diaphragm protein complex contains a group of scaffolding proteins that function to connect junctional membrane proteins to the actin cytoskeleton and signaling cascades. Despite their special morphology and function, there is considerable compositional similarity between the podocyte slit diaphragm and typical junctional complexes of other epithelial cells.

关键词： adhesion molecule   catenin   E-cadherin   IQGAP1   lung adenocarcinoma   micropapillary pattern  

摘要： Micropapillary differentiation in adenocarcinomas has recently been associated with poor prognosis because these tumors are more likely to metastasize. However, no clear explanation exists as to why the presence of a micropapillary pattern is associated with metastasis. A case of primary lung adenocarcinoma with a prominent micropapillary pattern is presented here, with special reference to the immunohistochemical expression of the E-cadherin-mediated system and IQGAP1. Histologically, the tumor was diagnosed as a moderately differentiated papillary adenocarcinoma, showing an extensive micropapillary pattern, with intrapulmonary metastases, pulmonary disseminations, lymphovascular invasions, and lymph node metastases. Immunohistochemically, positive staining for the adhesion molecules E-cadherin, alpha-catenin, and beta-catenin was detected in both the micropapillary and non-micropapillary areas, whereas IQGAP1 was detected in the micropapillary, but not in the non-micropapillary, area. The adhesive function of E-cadherin depends on the integrity of the entire cadherin-catenin-actin network, and thus the expression of IQGAP1 may lead to adherens junction disassembly, and consequently, the release of carcinoma cells organizing in a micropapillary pattern. This is the first report to suggest correlation between adenocarcinoma with a micropapillary pattern and the presence of adhesion molecules, and offers an intriguing first glimpse on the role of the micropapillary pattern in the process of metastasis.

关键词： Antigens   CD29   Cell Adhesion   Cell Membrane   Phosphoprotein Phosphatase  

摘要： Serine/threonine protein phosphatase (PP) 2A is thought to dephosphorylate phosphorylated beta 1 integrin to link with actin filaments (F-actin). However, whether PP2A participates in the regulation of F-actin assembly to which beta 1 integrin is anchored is unclear. We report here that the core enzyme of PP2A (PP2A-AC), consisting of the regulatory subunit A (PP2A-A) and the catalytic subunit C (PP2A-C), forms a complex with beta 1 integrin, a small GTPase Rac, and its effector IQGAP1 in non-malignant human mammary epithelial (HME) cells. Treatment of HME cells with okadaic acid (OA), in inhibitor of PP2A, caused cell rounding, reduction in F-actin assembly that links with beta 1 integrin, and dissociation of IQGAP1-bound PP2A-AC from Rac-beta 1 integrin. The dissociation of IQGAP1-PP2A-AC was accompanied by loss of F-actin gelating activity of Rac-beta 1 integrin. In breast cancer MCF-7 cells, which possess PP2A-C but lack PP2A-A, IQGAP1 was not associated with Rac-beta 1 integrin but with PP2A-C, with no distinct F-actin assembly that linked to Rac-beta 1 integrin even before treatment with OA, We therefore propose that PP2A, especially PP2A-A, functions to maintain F-actin assembly to which beta 1 integrin is anchored by recruitment of IQGAP1 to Rac-beta 1 integrin. (c) 2004 Wiley-Liss, Inc.

关键词： ACTOMYOSIN   CANCER treatment   GUANOSINE triphosphatase   BREAST cancer   PHOSPHOPROTEIN phosphatases  

摘要： Serine/threonine protein phosphatase (PP) 2A is thought to dephosphorylate phosphorylated 1 integrin to link with actin filaments (F-actin). However, whether PP2A participates in the regulation of F-actin assembly to which 1 integrin is anchored is unclear. We report here that the core enzyme of PP2A (PP2A-AC), consisting of the regulatory subunit A (PP2A-A) and the catalytic subunit C (PP2A-C), forms a complex with 1 integrin, a small GTPase Rac, and its effector IQGAP1 in non-malignant human mammary epithelial (HME) cells. Treatment of HME cells with okadaic acid (OA), an inhibitor of PP2A, caused cell rounding, reduction in F-actin assembly that links with 1 integrin, and dissociation of IQGAP1-bound PP2A-AC from Rac-1 integrin. The dissociation of IQGAP1-PP2A-AC was accompanied by loss of F-actin gelating activity of Rac-1 integrin. In breast cancer MCF-7 cells, which possess PP2A-C but lack PP2A-A, IQGAP1 was not associated with Rac-1 integrin but with PP2A-C, with no distinct F-actin assembly that linked to Rac-1 integrin even before treatment with OA. We therefore propose that PP2A, especially PP2A-A, functions to maintain F-actin assembly to which 1 integrin is anchored by recruitment of IQGAP1 to Rac-1 integrin. 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

关键词： IQGAP1   Rho-family GTPases   Rac1   Cdc42   cadherin   CLIP-170   APC   cell adhesion   cell polarization  

摘要： The dynamic rearrangement of cell-cell adhesion is one of the major physiological events in tissue development and tumor metastasis. Polarized cell migration, another key event, is a tightly regulated process that occurs during tissue development, chemotaxis and wound healing. Rho-family small GTPases, especially Rac1 and Cdc42, play pivotal roles in these processes through one of their effectors, IQGAP1. Recent studies reveal that IQGAP1 regulates cadherin-mediated cell-cell adhesion both positively and negatively. It captures and stabilizes microtubules through the microtubule binding protein CLIP-170 near the cell cortex, leading to establishment of polarized cell morphology and directional cell migration. Furthermore, Rac1 and Cdc42 link the adenomatous polyposis coli (APC) protein to actin filaments through IQGAP1 at the leading edge and thereby regulate polarization and directional migration.

关键词： PROTEIN-KINASE-C   ACTIN-BINDING   MASS-SPECTROMETRY   CELL-MIGRATION   SELECTIVEDETECTION   NEURONAL POLARITY   CDC42   CALMODULIN   CYTOSKELETON   GTPASES  

摘要： In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, beta-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase C epsilon, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a non-phosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.