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.

关键词： Biochemical engineering  

摘要： Circulating blood platelets regulate the initial phase of the hemostatic response through adhesive and aggregatory events and by providing the necessary procoagulant surface for prothrombinase complex assembly and thrombin generation. The signaling pathway(s) that regulate platelet procoagulant activity are largely unknown, although they are distinct from platelet aggregatory signals linked to fibrinogen ligation to the conformationally active alpha(IIB)beta(3) integrin. We describe a novel intracellular signaling mechanism involving platelet IQGAP1 that specifically regulates the development of platelet procoagulant activity under conditions of mechanical shear stress. Murine platelets that are deficient in IQGAP1 demonstrate increased prothrombinase activity compared with wild-type littermate controls when activated by a physiological shear stress of 16 dynes/cm(2) (shear rates of 1600 s(-1)) (p < 0.0001), corresponding to similar to 2.5 times the normal shear stress, or similar to 40% degree of stenosis in coronary arteries. The exaggerated prothrombinase activity is not associated with enhanced platelet microvesiculation ( cytoskeletal proteolysis) and occurs independently of the intracellular calcium release, [Ca2+](i), but it is specifically coupled to the alpha-granule exocytic pathway without concomitant effects on aminophospholipid exposure. These observations identify platelet IQGAP1 as an important modulator of normal hemostasis and as an appropriate pharmacological target for control of platelet procoagulant function.

关键词： calmodulin   IQGAP1   calcium   F-actin   Cdc42   Rac1  

摘要： IQGAPI regulates cytoskeletal dynamics through interactions with the Rho family GTPases Racl and Cdc42, F-actin, and beta-catenin. Calmodulin interaction with IQ mwotifs of IQGAPI negatively influences these IQGAPI interactions. Although, calmodulin interacts with IQGAPI in the absence of Ca2+ and Was suggested to exhibit reduced binding when Ca2(+) bound, recent reports show substantially greater binding when Ca2+ is present. Binding evaluations have primarily relied on IQGAPI interaction with calmodulin conjugated to Sepharose 4B. In this study we evaluated the Ca2+-dependence of calmodulin interaction with native IQGAPl using a series of independent biochemical approaches. We found the apparent binding of calmodulin to IQGAPI was Ca2+-independent. being between 5- and 20-fold greater in the absence than in the presence of Ca2+ lit addition, calmodulin in- teraction with IQGAPl was negatively regulated by buffer [Ca2-] (IC50 = 3.4 x 10(-7) M). Regulation was specific to Ca2+, as Ba2+ was similar to400-fold less effective than Ca2+ at modulating the interaction. Moreover, testing of calmodulin mutants demonstrated that apocalmodulin tightly binds IQGAPI and that the N- and C-terminal pair of EF hands are important for Ca2- sensitivity. These data indicate that calmodulin may disassemble from IQGAPI to facilitate IQGAPI interaction with effectors of cytoskeletal reorQanization durina conditions of cell activation that prornote increased cytosolic [Ca2+] (C) 2004 Elsevier Inc. All rights reserved.

关键词： Genetic engineering  

摘要： IQGAP1 binds several proteins including actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. The interaction with these targets enables IQGAP1 to participate in many cellular functions varying from regulation of the cytoskeleton to gene transcription. Here we show that extracellular signal-regulated kinase (ERK) 2 binds to IQGAP1. In vitro analysis with purified proteins demonstrated a direct interaction between ERK2 and IQGAP1. Moreover, binding occurred in cells as endogenous ERK2 co-immunoprecipitated with IQGAP1 from human breast epithelial cell lysates. The association between ERK2 and IQGAP1 was independent of epidermal growth factor. The in vivo interaction has functional significance. Manipulation of intracellular IQGAP1 levels significantly reduced growth factor-stimulated ERK1 and ERK2 activity. Similarly, stimulation of ERK1 and ERK2 activity by insulin-like growth factor I was reduced when IQGAP1 levels were changed. In contrast, overexpression of an IQGAP1 construct lacking the ERK2 binding region did not interfere with activation of ERK1 and ERK2 by epidermal growth factor. Our data disclose a previously unidentified communication between IQGAP1 and the ERK pathway and imply that IQGAP1 modulates the Ras/mitogen-activated protein kinase signaling cascade.

关键词： Biochemistry  

摘要： The dynamic processes of cell migration and invasion are largely coordinated by Rho family GTPases. The scaffolding protein IQGAP1 binds to Cdc42, increasing the amount of active Cdc42 both in vitro and in cells. Here we show that overexpression of IQGAP1 in mammalian cells enhances cell migration in a Cdc42- and Rac1-dependent manner. Importantly, cell motility was significantly decreased both by knock down of endogenous IQGAP1 using small interfering RNA and by transfection of a dominant negative IQGAP1 construct, IQGAP1DeltaGRD. Cell invasion was similarly altered by manipulating intracellular IQGAP1 concentrations. Moreover, invasion mediated by constitutively active Cdc42 was attenuated by IQGAP1DeltaGRD. Thus, IQGAP1 has a fundamental role in cell motility and invasion.

关键词： beta-cells   Rho family   GTPases   cAMP/PKA   IQGAP   signal transduct  

摘要： IQGAP1, is a recently discovered scaffold protein proposed to regulate membrane cytoskeleton events through protein-protein interactions with F-actin, E-cadherin, beta-catenin, and CLIP170. The binding of IQGAP1 to its partners is regulated by calcium/calmodulin (Ca++/CaM) and the small molecular weight guanine nucleotide triphosphatases (GTPases), Cdc42, and Rac1. Here we identify a novel IQGAP1 scaffolding function by isolating the cyclic AMP dependent kinase (PKA) with IQGAP1. IQGAP1 was co-purified with PKA using 5'-cyclic AMP (cAMP) affinity chromatography and PKA activity was co-immunoprecipitated with IQGAP1 using an anti-IQGAP1 antibody. The association of IQGAP1 with PKA was shown to Occur through a direct interaction between A kinase anchoring protein 79 (AKAP79) and the carboxyl-terminal domain of IQGAP1. This suggests that cAMP/PKA may be coupled with Ca++/CaM and GTPases through an IQGAP1/AKAP79 complex.

关键词： IQGAP1   cdc42   Rho GTPase   cytoskeleton  

摘要： IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP11 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1. (C) 2003 Elsevier Science (USA). All rights reserved.

关键词： cytoskeleton   rho GTPase   calcium   calmodulin   cytokinesis  

摘要： A family of proteins known as IQGAPs have been identified in yeast, amebas and mammals. IQGAPs are multidomain molecules that contain several protein-interacting motifs which mediate binding to target proteins. Mammalian IQGAP1 is a component of signaling networks that are integral to maintaining cytoskeletal architecture and cell-cell adhesion. Published data suggest that IQGAP1 is a scaffolding protein that modulates cross-talk among diverse pathways in complex regulatory circuits. These pathways include modulating the actin cytoskeleton, mediating signaling by Rho family GTPases and calmodulin, regulating E-cadherin and beta-catenin function and organizing microtubules. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

关键词： nectin   IQGAP1   cadherin   adherens junctions   actin  

摘要： IQGAP1, a putative downstream target of the Rho family small G proteins, Cdc42 and Rac, localizes at adherens junctions (AJs) in epithelial cells. It has been suggested that IQGAP1 localizes at AJs through its binding to beta-catenin, and negatively regulates the E-cadherin-mediated cell-cell adhesion. Nectin is a Ca2+-independent, immunoglobulin-like cell-cell adhesion molecule that localizes at AJs. Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs cooperatively with E-cadherin. Here we investigated a role of nectin in the localization of IQGAP1 at AJs. Ca2+ chelation from the medium causes disruption of the E-cadherin-mediated cell-cell adhesion, but not the nectin-based cell-cell adhesion, in Madin-Darby canine kidney (MDCK) cells. IQGAP1 remained at the residual nectin-based cell-cell adhesion sites where the E-cadherin immunofluorescence signal disappeared. Restoration of Ca2+ in the medium causes re-accumulation of E-cadherin to the residual pectin-based cell-cell adhesion sites to reform AJs. Nectin inhibitors inhibit this re-accumulation of E-cadherin to re-form AJs by impairing the pectin-based cell-cell adhesion. The nectin inhibitors also reduced the localization of IQGAP1 at the cell-cell adhesion sites. When MDCK cells were incubated with microbeads coated with the extracellular fragment of nectin that interacts with cellular nectin, IQGAP1 also accumulated at the bead-MDCK cell contact sites. The accumulation of IQGAP1 at the cell-cell adhesion sites was inhibited by actin filament-disrupting agents, latrunculin A and cytochalasin D. These results indicate that nectin is involved in the localization of IQGAP1 at AJs through the actin cytoskeleton.

关键词： autoimmunity   cellular structure   epidermal proteins   subepidermal blistering  

摘要： Autoantibodies in patients with autoimmune bullous skin diseases, such as pemphigus or bullous pemphigoid are of diagnostic value and might play a part in the pathogenic scenario. In this study we present five patients with erythematous plaques, subepidermal blister formation of the skin, and the presence of circulating autoantibodies directed against a so far unrecognized 190 kDa antigen in human keratinocytes. Amino acid sequence analysis identified the protein as IQGAP1, a recently described human Ras GTPase-activating-like protein suspected to act as an effector molecule for Cdc42 and Rac1, members of the Rho small GTPase family and to play a key part in regulating E-cadherin-mediated cell adhesion. The protein is selectively recognized by a monoclonal anti-IQGAP1 antibody on western blots and immunoprecipitates from keratinocyte extracts. Indirect immunofluorescence locates IQGAP1 within individual keratinocytes in a cytoplasmic pattern and along the cell periphery at adhesive sites. Our results demonstrate IQGAP1, a newly described multifunctional protein, to be constitutively expressed in human keratinocytes where it may contribute to the integrity of the epidermal layer. Furthermore, we found autoantibodies reacting with IQGAP1 in patients with bullous skin eruptions most apparently belonging to the spectrum of bullous pemphigoid.