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： Human IQGAP1 is a widely expressed 190-kDa Cdc42-, Rac1-, and calmodulin-binding protein that interacts with F-actin in vivo and that can cross-link F-actin microfilaments in vitro. Recent results have implicated IQGAP1 as a component of pathways via which Cdc42 or Rad modulates cadherin-based cell adhesion (S, Kuroda et al,, Science 281:832-835, 1998), whereas yeast IQGAP-related proteins have been found to play essential roles during cytokinesis. To identify critical in vivo functions of IQGAP1, we generated deficient mice by gene targeting. We demonstrate that IQGAP1 null mutants arise at normal frequency and show no obvious defects during development or for most of their adult life. Loss of IQGAP1 also does not affect tumor development or tumor progression, but mutant mice exhibit a significant (P < 0.0001) increase in late-onset gastric hyperplasia relative to wild-type animals of the same genetic background. While we cannot exclude that functional redundancy with IQGAP2 contributes to the Lack of developmental phenotypes, the restricted expression pattern of IQGAP2 is not obviously altered in adult IQGAP1 mutant mice. Thus, IQGAP1 does not serve any essential nonredundant functions during murine development but may serve to maintain the integrity of the gastric mucosa in older animals.

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.

关键词： cell adhesion   Rho   GTPase   catenin   cadherin   integrin   Rho-kinase   IQGAP1  

摘要： The Rho small GTPases, Cdc42, Ra1 and Rho, are implicated in regulation of integrin-mediated cell-substratum adhesion and cadherin-mediated cell-cell adhesion. Identification and characterization of effectors of these GTPases have provided insights into their modes of action. Rho-kinase, an effector of Rho, regulates integrin-mediated cell-substratum adhesion (focal adhesion) by regulating the phosphorylation state of myosin light chain (MLC): it directly phosphorylates MLC and also inactivates myosin phosphatase, IQGAP1, an effector of Cdc42 and Ra1, regulates cadherin-mediated cell-cell adhesion by interacting with beta-catenin and dissociating alpha-catenin from the cadherin-catenins complex. Activated Cdc4.2 and Rad inhibit IQGAP1, thereby stabilizing the cadherin-catenins complex. Cdc42/Rac1 and IQGAP1 thus appear to constitute a switch that regulates cadherin-mediated cell-cell adhesion.

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.