关键词： IQGAP1   E-cadherin   catenin   gastric cancer   immuno-histochemistry  

摘要： Down-regulation of E-cadherin function is characteristic of cancer cells and might involve the small C-protein Rho family, including pac I and Cdc42, IQGAP I has been reported to be one of the target proteins of pac I and Cdc42, To elucidate the role of IQGAPI in cancer-cell adhesion, its expression was investigated in 47 cases of human gastric cancer by immunohistochemistry and Western blot upon protein fractionation, especially in comparison with E-cadherin and catenin expression. In the non-cancerous columnar epithelium of the stomach, IQGAPI, as well as E-cadherin/catenin, was expressed at the cell-cell boundary. IQGAP I was frequently observed diffusely in the cytoplasm in intestinal-type tumors (20/22 cases) but was expressed at the cell membrane in diffuse-type tumors (19/25 cases), thus showing significant association with tumor differentiation (p, < 0.01). Interestingly, membranous expression of IQGAP I was inversely correlated with that of E-cadherin ( < 0.05) or alpha -catenin (p < 0.001), These observations were consistent with the Western blot results following protein fractionation. IQGAPI was dominantly expressed in the soluble fraction in differentiated tumors;however, in undifferentiated tumors, it was mostly in the insoluble fraction. In contrast, both E-cadherin and -catenin were detected only in the insoluble fraction. Thus, subcellular localization of IQGAP I from the cytoplasm to the cell membrane was correlated with E-cadherin dysfunction and tumor dedifferentiation in gastric carcinogenesis. (C) 2001 Wiley-Liss, Inc.

关键词： gastric cancer   15q26   gene amplification   IQGAP1   beta-catenin  

摘要： Our previous comparative genomic hybridization (CGH) study revealed a novel amplified region at 15q26 in two cell lines established from diffuse types of gastric cancer (GC). In this amplified region, FES and IGF1R, known targets on 15q26, were located telomeric to the amplicon in the two cell lines, HSC39 and 40A, suggesting that another tumor-associated gene exists in this region. While screening expressed sequence tags (ESTs) for novel genes in this region, we identified the IQGAP1 amplification. IQGAP1 has been reported to encode a ras GAP-related protein, and its interaction with cadherin and/or beta -catenin induces a dissociation of beta -catenin from the cadherin-catenin complex. one of the mechanisms for cell-cell adhesion. Northern and Western blot analyses revealed that amplification of this gene was accompanied by corresponding increases in mRNA and protein expression. Moreover, immunocytochemical staining showed that overexpressed IQGAP1 accumulated at the membrane, suggesting its colocalization with beta -catenin. Taken together, these findings suggest that IQGAP1 may be one of the target genes in the 15q26 amplicon correlated with a malignant phenotype of gastric cancer cells, such as diffuse and invasive characteristics, through the disruption of E-cadherin-mediated cell-cell adhesion.

关键词： glioma   gene array   cadherin   catenin   IQGAP1   RhoA  

摘要： Transforming growth factor-alpha (TGF-alpha) and its receptor are frequently co-expressed in high-grade astrocytomas, suggesting a role for TCF-alpha autocrine/paracrine loops in the malignant progression of astrocytomas. To identify genes that may be critical in mediating TGF-alpha impact on the malignant progression of astrocytomas, we have used cDNA arrays to investigate TGF-alpha effects on the gene expression profile of U-373 MG glioblastoma cells. We found that in these cells similar to 50% of the TGF-alpha regulated genes code for cell motility/invasion-related proteins. TGF-alpha action on the expression of four of these proteins, alpha -catenin, IQGAP1, RhoA, and cadherin-ll, was further investigated by immunoblotting in four astrocytoma cell lines and in normal astrocytes. The results demonstrate that the effects of TGF-alpha on IQGAP1, alpha -catenin, and RhoA expression are cell-line dependent. On the other hand, under TGF-alpha treatment, cadherin-li expression is consistently decreased in all astrocytoma cell lines tested but is increased in normal astrocytes. In addition, we found that cadherin-ll is consistently down-regulated in astrocytomas versus normal brain tissues. Altogether, these results suggest that the down-regulation of cadherin-ll is a frequent molecular event in the neoplastic transformation of astrocytes and that this down-regulation may be initiated and/or amplified by TGF-alpha autocrine/paracrine loops during tumor progression.

关键词： IQGAP1   alpha-catenin   E-cadherin   endometrial cancer  

摘要： E-cadherin function is thought to be impaired in epithelial cancer. To investigate the alterations in E-cadherin associated with cytoplasmic molecules including alpha -catenin, beta -catenin, gamma -catenin, p120CAS, and IQGAP1 in various endometrial cancers with different degree of differentiation, we examined the localization and expression of E-cadherin and cytoplasmic molecules in 30 cases of both well and poorly differentiated endometrioid adenocarcinomas, using immunofluorescence and immunoblotting techniques. E-cadherin and cytoplasmic molecules demonstrated linear staining at the cell boundaries in normal endometrium. In all 20 cases with well differentiated adenocarcinomas, alpha -catenin and IQGAP1 disappeared from the cell adhesive sites, but other cytoplasmic molecules were co-localized with E-cadherin along the cell boundaries. In all 20 cases with poorly differentiated adenocarcinomas, E-cadherin and cytoplasmic molecules accumulated as large aggregates along cell adhesive sites, and the localization of IQGAP1 differed from those of other cytoplasmic molecules, The expression of these molecules in all 20 cases with well differentiated adenocarcinomas decreased or was lost in Triton-insoluble fraction, in comparison with the findings for all cases with normal endometrium or poorly differentiated adenocarcinomas, These results suggested that each alteration in E-cadherin associated with cytoplasmic molecules may play a different role in E-cadherin dysfunction between well and poorly differentiated adenocarcinomas. (C) 2000 Cancer Research Campaign.

摘要： Ca2+-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin, Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion, On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.

摘要： The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization, Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1, To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector pal-binding domain (PBD) interaction, Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rad, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 anal 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/ RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP- binding activity could retain the peripheral. actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction, Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.

摘要： IQGAP1, a target of Cdc42 and Bad small GTPases, directly interacts with beta-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating alpha-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, R., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I,, Matsuura, Y., Yonehara, S,, and Kaibuchi, IT. (1998) Science 281, 832-855). Here we investigated how Cdc42 and Rad regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1-183) of beta-catenin, which contains the alpha-catenin-binding domain. IQGAP1 dissociated alpha-catenin from the beta-catenin-alpha-catenin complex in a dose-dependent manner in vitro. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) glutathione S-transferase (GST)Cdc42 and GTP gamma ***-Rac1 inhibited the binding of IQGAP1 to beta-catenin in a dose-dependent manner vitro, whereas neither ***-Cdc42, ***-Rac1, in nor GTP gamma S GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of alpha-catenin from the cadherin-catenin complex induced by the overexpression of IQGAP1 in L cells expressing E-cadherin (EL cells). Consistent with this, the overexpression of either dominant negative Cdc42 or Rad resulted in the reduction of E-cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rad negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with beta-catenin, leading to stabilization of the cadherin-catenin complex.

摘要： Cell-cell adhesion is a dynamic process in various cellular and developmental situations. Cadherins, well-known Ca2+-dependent adhesion molecules, are thought to play a major role in the regulation of cell-cell adhesion. However, the molecular mechanism underlying the rearrangement of cadherin-mediated cell-cell adhesion is largely unknown. Cdc42 and Rad, belonging to the Rho small GTPase family, have recently been shown to be involved in the regulation of cell-cell adhesion. In addition, IQGAP1, an effector for Cdc42 and Rad, has been shown to regulate the cadherin function through interaction with beta-catenin, a molecule associated with cadherin. In this review, we will summarize the mode of action of Cdc42 and Rad as well as IQGAP1 as molecular switches for the cadherin function, and then discuss physiological processes in which the Cdc42/Rac1/IQGAP1 system may be involved. (C) 1999 Academic Press.

摘要： Calmodulin regulates diverse Ca2+-dependent cellular processes, including cell cycle progression and cytoskeletal rearrangement. A recently identified calmodulin-binding protein, IQGAP1, interacts with both actin and Cdc42, In this study, evidence is presented that, in the absence of Ca2+, IQGAP1 bound to Cdc42, which maintained Cdc42 in the active GTP-bound state. Addition of Ca2+ both directly abrogated the effect of IQGAP1 on the intrinsic GTPase activity of Cdc42 and, in the presence of calmodulin, dissociated Cdc42 from IQGAP1. In addition, in vitro binding assays revealed that calmodulin associated with both the calponin homology domain and the IQ motifs of IQGAP1. Moreover, F-actin competed with Ca2+/calmodulin for binding to the calponin homology domain, but not the IQ motifs, of IQGAP1, Analysis of cell lysates revealed that calmodulin bound to IQGAP1 in a ternary complex with Cdc42, Increasing the Ca2+ concentration enhanced the interaction between calmodulin and IQGAP1, with a concomitant decrease in the association of IQGAP1 with Cdc42. Our data suggest that IQGAP1 functions as a scaffolding protein, providing a molecular link between Ca2+/calmodulin and Cdc42 signaling.

摘要： The 190 kD human IQGAP1 protein, by virtue of its N-terminal calponin-homology domain, is found associated with the actin cytoskeleton, and is capable of cross-linking actin filaments. IQGAP1 complexes with several proteins, including the Rho family GTPases Cdc42 and Rac, as well as calmodulin. It was previously noted that one of the IQ motifs of IQGAP1 displays significant similarity to a myosin heavy chain IQ motif responsible for binding the calmodulin-related myosin essential light chain (ELC). Employing the yeast two-hybrid methodology as well as in vitro binding experiments, we present evidence that a truncated version of IQGAP1 can interact with the myosin ELC. This interaction may have significant consequences for various cellular processes that involve actomyosin contractility, and suggests that the biological targets of the ELC may not be restricted to the myosin heavy chain. (C) 1998 Academic Press.