关键词： TUMOR-CELL-MIGRATION   LYMPHOMA TRANSMEMBRANE GLYCOPROTEIN   CD44 INTERACTION   ESTROGEN-RECEPTOR   PROTEIN-KINASE   CYCLIN D1   NUCLEAR TRANSLOCATION   MESOTHELIAL CELLS   REGULATEDKINASE   RNA INTERFERENCE  

摘要： In this study, we have examined the interaction of hyaluronan (HA)-CD44 with IQGAP1 (one of the binding partners for the Rho GTPase Cdc42) in *** human ovarian tumor cells. Immunological and biochemical analyses indicated that IQGAP1 (molecular mass of similar to 190 kDa) is expressed in *** cells and that IQGAP1 interacts directly with Cdc42 in a GTP-dependent manner. Both IQGAP1 and Cdc42 were physically linked to CD44 in *** cells following HA stimulation. Furthermore, the HA-CD44-induced Cdc42-IQGAP1 complex regulated cytoskeletal function via a close association with F-actin that led to ovarian tumor cell migration. In addition, the binding of HA to CD44 promoted the association of ERK2 with the IQGAP1 molecule, which stimulated both ERK2 phosphorylation and kinase activity. The activated ERK2 then increased the phosphorylation of both Elk-1 and estrogen receptor-alpha (ER alpha), resulting in Elk-1- and estrogen-responsive element-mediated transcriptional up-regulation. Down-regulation of IQGAP1 (by treating cells with IQGAP1-specific small interfering RNAs) not only blocked IQGAP1 association with CD44, Cdc42, F-actin, and ERK2 but also abrogated HA-CD44-induced cytoskeletal function, ERK2 signaling (e.g. ERK2 phosphorylation/activity, ERK2-mediated Elk-1/ER alpha phosphorylation, and Elk-1/ER alpha-specific transcriptional activation), and tumor cell migration. Taken together, these findings indicate that HA-CD44 interaction with IQGAP1 serves as a signal integrator by modulating Cdc42 cytoskeletal function, mediating Elk-1-specific transcriptional activation, and coordinating "cross-talk" between a membrane receptor ( CD44) and a nuclear hormone receptor (ER alpha) signaling pathway during ovarian cancer progression.

关键词： Intestinal Neoplasms   Mutation   Stomach Neoplasms   Variation (Genetics)  

摘要： IQGAP1 is recognized as a negative regulator of cell-cell adhesion at adherens junctions in several cell types, including gastric mucosal cells. The histopathologic appearance of diffuse gastric carcinoma is defined by non- or poorly cohesive tumor cells, indicating abnormal intercellular adhesion. Hence, we screened 38 gastric cancers for activating point mutations in IQGAP1. In 2 of the 33 diffuse gastric cancers, there was a missense nucleotide change predicted to alter the amino acid sequence in the GAP-related domain, which includes part of the binding site for the activated small G proteins Cdc42 and Rac1. Many intronic IQGAP1 gene changes were observed, and several occurred more frequently in diffuse-type gastric cancers than in intestinal-type gastric cancers. A highly variable pentanucleotide repeat was identified in the final intron of IQGAP1. The most expanded six-repeat sequence was present exclusively in diffuse-type gastric cancers. Additionally, 19 diffuse cases and two intestinal cases exhibited silent coding region nucleotide alterations. Taken together, our results suggest that IQGAP1 coding sequence mutations are not a frequent event in gastric cancer, but do occur in a subset of diffuse-type gastric carcinomas. Additional studies analyzing other proteins involved in cell adhesion may lead to a better molecular understanding of the histopathologic appearance of diffuse gastric cancers. (C) 2004 Wiley-Liss, Inc.

关键词： protein phosphatase 2A   beta 1-integrin   Rae   IQGAP1   F-actin assembly   cell adhesion  

摘要： Assembly of F-actin that links with beta1-integrin during the G1 phase of cell cycle is released from beta1-integrin and disrupted at mitosis. However, it remains unclear how F-actin assembly to which beta1-integrin anchors is cell cycle-dependently regulated. We show that beta1-integrin was co-immunoprecipitated and co-localized with a small GTPase Rac and its effector IQGAP1, along with PP2A-AC, in HME cells during G1. When the cells were accumulated to G2/M, the co-immunoprecipitation or co-localization of IQGAP1 and PP2A-AC with beta1-integrin was lost, leaving Rac bound to beta1-integrin. The dissociated IQGAP1 was co-immunoprecipitated with the concomitantly dissociated PP2A-A and -C, indicating the complex formation among the proteins in G2/M cells. Falling ball viscometric assays revealed that only IQGAP1-bound beta1-integrin-Rac in G1 cells exhibited an enhanced F-actin cross-linking activity. The results suggest that the mitotic loss of F-actin assembly to which beta1-integrin anchors is due to PP2A-mediated dissociation of IQGAP1 from Rac-bound beta1-integrin. (C) 2004 Elsevier Inc. All rights reserved.

关键词： Fyn   IQGAP1   phosphoinositide 3-kinase   podocyte   slit diaphragm  

摘要： Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.

关键词： Adherens Junctions   Cadherins   Germ Cells   Sertoli Cells   cdc42 GTP-BindingProtein  

摘要： The movement of developing germ cells across the seminiferous epithelium during spermatogenesis involves extensive adherens junction (AJ) restructuring between Sertoli cells, as well as between Sertoli and germ cells. In this report, we show that the intricate interactions between Cdc42 (a Rho family protein of Mr similar to23 kDa originally identified in membranes of human platelets and placenta, and is the homolog of CDC42Sc, which is known to regulate of bud-site assembly in Saccharomyces cerevisiae) and its effector, (IQ) under bar motif containing (G) under bar TPase activating protein (IQGAP1, Mr similar to189 kDa, it is also an actin-binding protein known to interact with Cdc42 and Rac1 GTPases), regulate Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics. Using testis lysates for immunoprecipitation (IP), IQGAP1 was shown to associate with E-cadherin, N-cadherin, and beta-catenin (but not beta1-integrin and nectin-2), as well as with actin and vimentin (but not pi-tubulin). Moreover, IQGAP1 was found to localize to the periphery of both Sertoli and germ cells in the seminiferous epithelium, at sites of cell-cell contacts. Using fluorescent microscopy with dual fluorescent probes, IQGAP1 was found to co-localize, at least in part, with N-cadherin in the seminiferous epithelium consistent with their localization at the basal and apical ES. Using Sertoli-germ cell cocultures, it was demonstrated that AJ assembly associated with a transient induction of Cdc42 and IQGAP1, which was not found when Sertoli cells were cultured alone. Lastly, a shift in the interactions of Cdc42, IQGAP1, beta-catenin, and N-cadherin was detected in Sertoli-germ cell cocultures using an C2+-induced AJ disruption model, which was used to examine AJ disassembly and its reassembly. In the presence of Ca2+ IQGAP1 bound preferentially to Cdc42 rather than to P-catenin. However, when Ca2+ was depleted from cocultures using EGTA, a Ca2+ chelating agent, IQGAP1 lost its affinity for

关键词： Actins   Adenomatous Polyposis Coli Protein   Animals   Binding Sites   COS Cells   Cell Movement   Cercopithecus aethiops   Fibroblasts   Glutathione Transferase   Green Fluorescent Proteins   Image Processing   Computer-Assisted   Immunoprecipitation   Microscopy   Fluorescence   Microtubule-Associated Proteins   Microtubules   Models   Biological   Neoplasm Proteins   Plasmids   Protein Binding   RNA   Small Interfering   Recombinant Fusion Proteins   Research Support   Non-U.S. Gov't   Signal Transduction   Time Factors   Transfection   Vero Cells   Wound Healing   cdc42 GTP-Binding Protein   rac1 GTP-Binding Protein   ras GTPase-Activating Proteins  

摘要： Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.

关键词： Cells   Phosphorylation   GTP Phosphohydrolases   binding  

摘要： The Rho-GTPase Cdc42 is important for the establishment and maintenance of epithelial polarity. Signaling from Cdc42 is propagated via its effector molecules that specifically bind to Cdc42 in the GTP-bound form. The cell-cell contact regulator and actin-binding protein IQGAP1 is described as effector of Cdc42 and Rac. Unexpectedly, we show in this study that IQGAP1 bound also directly nucleotide-depleted Cdc42 (Cdc42-ND). This interaction was enhanced in the presence of phosphatase inhibitors and in epithelial cells without cell-cell contacts. Tandem mass spectrometry analysis and immunoprecipitation experiments revealed that IQGAP1 was Ser(1443)-phosphorylated in vivo, potentially by protein kinase Cepsilon and upon loss of cell-cell contacts. In addition, we identified two independent domains of the IQGAP1 C terminus that bound exclusively Cdc42-ND. These domains interacted with each other, favoring the binding to Cdc42-GTP. Moreover, phosphorylation on Ser(1443) strongly inhibited this intramolecular interaction. Thus, we unraveled a molecular mechanism that reveals a novel type of Rho-GTPase regulator. We propose that, depending on its phosphorylation state, IQGAP1 might serve as an effector or sequester nucleotide-free Cdc42 to prevent signaling.

关键词： cell motility   lammelipodium   Cdc42   Rac1   calmodulin  

摘要： IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, beta-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5-50 muM K-d) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP1(2-210), which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP1(2-210) was found to be monomeric, to bind F-actin with a K-d of similar to47 muM, to saturate F-actin at a molar ratio of one IQGAP1(2-210) per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. (C) 2004 Wiley-Liss, Inc.

关键词： cell-free assay   E-cadherin   endocytosis   Rho family small G proteins   IQGAP1  

摘要： E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.