关键词： Biochemistry  

摘要： IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and beta-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca2+ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAPI much more efficiently in the presence of Ca2+ than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca2+ reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in f ree intracellular Ca2+ promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments.

关键词： Biochemistry  

摘要： beta-Catenin, an oncoprotein integral to cell-cell adhesion and proliferative signaling, is increased in several malignancies. The recently discovered calmodulin-binding protein IQGAP1 binds stoichiometrically to beta-catenin and regulates the association of beta-catenin with the cell-cell adhesion complex. In the present work, we investigated the role of IQGAP1 on the transcriptional co-activator function of beta-catenin, and whether calmodulin modulated the functional interaction between IQGAP1 and beta-catenin. In vitro competition assays revealed that both Ca2+/calmodulin and apo-calmodulin, when pre-bound to IQGAP1, prevented binding of beta-catenin to IQGAP1, and partially displaced beta-catenin pre-bound to IQGAP1 when added subsequently. Conversely, beta-catenin partially displaced apo-calmodulin, but not Ca2+/calmodulin, from IQGAP1. Overexpression of IQGAP1 in SW480 colon carcinoma cells enhanced beta-catenin-mediated transcriptional co-activation by 1.72-fold, and this stimulation was significantly attenuated upon antagonism of calmodulin using the cell-permeable antagonist CGS9343B. Moreover, an IQGAP1 mutant that does not bind calmodulin was unable to stimulate beta-catenin transcriptional function. Results of pulse-chase analyses suggested that IQGAP1 slowed the turnover of soluble, but not total, beta-catenin. Immunocytochemistry revealed that IQGAP1 overexpression increased the amount of beta-catenin located in the nucleus, whereas incubation of cells with CGS9343B blocked this accumulation. Together, our results imply that IQGAP1 enhances the function of beta-catenin in the nucleus and that calmodulin regulates this stimulation.

关键词： IQGAP1   colorectal cancer   cell migration   invasion   cell-cell adhesion   immunohistochemistry  

摘要： In histopathological sections, it is frequently observed that carcinoma cells move in and invade the stroma as coherent cell nests, rather than single cells. We have called this type of movement 'cohort migration (CM)' and developed an in vitro model, in which human colon adenocarcinoma cells move as coherent cell sheets when stimulated with naturally occurring motogenic factor, hepatocyte growth factor/scatter factor (HGF/SF). In this CM model, localized release from cell-cell adhesion is essential for cell movement. Recently, we have shown that IQGAP1, a target molecule of Cdc42 and Rac1 small GTPases, is involved in this localized release from the E-cadherin-based cell-cell adhesion during CM. In this study, we examined expression of IQGAP1 immunohistochemically in human colorectal tissues and found that IQGAP1 was overexpressed in carcinoma tissues compared with normal counterparts. Within the carcinoma tissue, IQGAP1 tended to be expressed more at the invasion front than at the upper portions, and higher levels of expression were observed in deeper two-thirds of carcinoma tissues than in the superficial one-third. This expression pattern showing stronger signals in deeper portions was most apparent in advanced carcinomas that invaded into the subserosa. These findings supported a role of IQGAP1 in colon carcinoma invasion. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

关键词： Proteins  

摘要： IQGAP1 contains a number of protein recognition motifs through which it binds to targets. Several in vitro studies have documented that IQGAP1 interacts directly with calmodulin, actin, E-cadherin, beta -catenin, and the small GTPases Cdc42 and Rac. Nevertheless, direct demonstration of in vivo function of mammalian IQGAP1 is limited. Using a novel assay to evaluate in vivo function of IQGAP1, we document here that microinjection of IQGAP1 into early Xenopus embryos generates superficial ectoderm. lesions at late blastula stages. This activity was retained by the mutated variants of IQGAP1 in which the calponin homology domain or the WW domain was deleted. By contrast, deletion of the IQ (IQGAP1-Delta IQ), Ras-GAP-related (IQGAP1-Delta GRD), or C-terminal (IQGAP1-DeltaC) domains abrogated the effect of IQGAP1 on the embryos. None of the latter mutants bound Cdc42, suggesting that the binding of Cdc42 by IQGAP1 is critical for its function. Moreover, overexpression of IQGAP1, but not IQGAP1-Delta GRD, significantly increased the amount of active Cdc42 in embryonic cells. Co-injection of wild type IQGAP1 with dominant negative Cdc42, but not the dominant negative forms of Rac or Rho, blocked the effect of IQGAP1 on embryonic ectoderm. Together these data indicate that the activity of IQGAP1 in embryonic ectoderm. requires Cdc42 function.

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.