共查询到20条相似文献,搜索用时 49 毫秒
1.
Guichard C Pedruzzi E Dewas C Fay M Pouzet C Bens M Vandewalle A Ogier-Denis E Gougerot-Pocidalo MA Elbim C 《The Journal of biological chemistry》2005,280(44):37021-37032
The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b(558), the cytosol factors p47(phox), p67(phox), p40(phox), and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47(phox), as well as the recruitment of flavocytochrome b(558), p47(phox), and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b(558), p47(phox), or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67(phox) into detergent-resistant microdomains. Moreover, methyl-beta-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils. 相似文献
2.
Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly 总被引:5,自引:0,他引:5
Gorzalczany Y Sigal N Itan M Lotan O Pick E 《The Journal of biological chemistry》2000,275(51):40073-40081
The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559). 相似文献
3.
The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b558, in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase. 相似文献
4.
RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells 总被引:1,自引:0,他引:1
El-Sibai M Pertz O Pang H Yip SC Lorenz M Symons M Condeelis JS Hahn KM Backer JM 《Experimental cell research》2008,314(7):1540-1552
Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42. 相似文献
5.
Wojciak-Stothard B Tsang LY Haworth SG 《American journal of physiology. Lung cellular and molecular physiology》2005,288(4):L749-L760
Hypoxia/reoxygenation-induced changes in endothelial permeability are accompanied by endothelial actin cytoskeletal and adherens junction remodeling, but the mechanisms involved are uncertain. We therefore measured the activities of the Rho GTPases Rac1, RhoA, and Cdc42 during hypoxia/reoxygenation and correlated them with changes in endothelial permeability, remodeling of the actin cytoskeleton and adherens junctions, and production of ROS. Dominant negative forms of Rho GTPases were introduced into cells by adenoviral gene transfer and transfection, and inhibitors of NADPH oxidase, PI3 kinase, and Rho kinase were used to characterize the signaling pathways involved. In some experiments constitutively activated forms of RhoA and Rac1 were also used. We show for the first time that hypoxia/reoxygenation-induced changes in endothelial permeability result from coordinated actions of the Rho GTPases Rac1 and RhoA. Rac1 and RhoA rapidly respond to changes in oxygen tension, and their activity depends on NADPH oxidase- and PI3 kinase-dependent production of ROS. Rac1 acts upstream of RhoA, and its transient inhibition by acute hypoxia leads to activation of RhoA followed by stress fiber formation, dispersion of adherens junctions, and increased endothelial permeability. Reoxygenation strongly activates Rac1 and restores cortical localization of F-actin and VE-cadherin. This effect is a result of Rac1-mediated inhibition of RhoA and can be prevented by activators of RhoA, L63RhoA, and lysophosphatidic acid. Cdc42 activation follows the RhoA pattern of activation but has no effect on actin remodeling, junctional integrity, or endothelial permeability. Our results show that Rho GTPases act as mediators coupling cellular redox state to endothelial function. 相似文献
6.
Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases 总被引:28,自引:0,他引:28
A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versus RhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60 cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities. 相似文献
7.
Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation. 相似文献
8.
Recognition of cognate Rho GTPases by guanine-nucleotide exchange factors (GEF) is fundamental to Rho GTPase signaling specificity. Two main GEF families use either the Dbl homology (DH) or the DOCK homology region 2 (DHR-2) catalytic domain. How DHR-2-containing GEFs distinguish between the GTPases Rac and Cdc42 is not known. To determine how these GEFs specifically recognize the two Rho GTPases, we studied the amino acid sequences in Rac2 and Cdc42 that are crucial for activation by DOCK2, a Rac-specific GEF, and DOCK9, a distantly related Cdc42-specific GEF. Two elements in the N-terminal regions of Rac2 and Cdc42 were found to be essential for specific interactions with DOCK2 and DOCK9. One element consists of divergent amino acid residues in the switch 1 regions of the GTPases. Significantly, these residues were also found to be important for GTPase recognition by Rac-specific DOCK180, DOCK3, and DOCK4 GEFs. These findings were unexpected because the same residues were shown previously to interact with GTPase effectors rather than GEFs. The other element comprises divergent residues in the beta3 strand that are known to mediate specific recognition by DH domain containing GEFs. Remarkably, Rac2-to-Cdc42 substitutions of four of these residues were sufficient for Rac2 to be specifically activated by DOCK9. Thus, DOCK2 and DOCK9 specifically recognize Rac2 and Cdc42 through their switch 1 as well as beta2-beta3 regions and the mode of recognition via switch 1 appears to be conserved among diverse Rac-specific DHR-2 GEFs. 相似文献
9.
Arabidopsis RopGAPs are a novel family of rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for rop-specific GTPase stimulation 总被引:7,自引:0,他引:7
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The plant-specific Rop subfamily of Rho GTPases, most closely related to the mammalian Cdc42 and Rac GTPases, plays an important role in the regulation of calcium-dependent pollen tube growth, H(2)O(2)-mediated cell death, and many other processes in plants. In a search for Rop interactors using the two-hybrid method, we identified a family of Rho GTPase-activating proteins (GAP) from Arabidopsis, termed RopGAPs. In addition to a GAP catalytic domain, RopGAPs contain a Cdc42/Rac-interactive binding (CRIB) motif known to allow Cdc42/Rac effector proteins to bind activated Cdc42/Rac. This novel combination of a GAP domain with a CRIB motif is widespread in higher plants and is unique to the regulation of the Rop GTPase. A critical role for CRIB in the regulation of in vitro RopGAP activity was demonstrated using point and deletion mutations. Both types of mutants have drastically reduced capacities to stimulate the intrinsic Rop GTPase activity and to bind Rop. Furthermore, RopGAPs preferentially stimulate the GTPase activity of Rop, but not Cdc42 in a CRIB-dependent manner. In vitro binding assays show that the RopGAP CRIB domain interacts with GTP- and GDP-bound forms of Rop, as well as the transitional state of Rop mimicked by aluminum fluoride. The CRIB domain also promotes the association of the GAP domain with the GDP-bound Rop, as does aluminum fluoride. These results reveal a novel CRIB-dependent mechanism for the regulation of the plant-specific family of Rho GAPs. We propose that the CRIB domain facilitates the formation of or enhanced GAP-mediated stabilization of the transitional state of the Rop GTPase. 相似文献
10.
Burritt JB Foubert TR Baniulis D Lord CI Taylor RM Mills JS Baughan TD Roos D Parkos CA Jesaitis AJ 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(12):6082-6089
mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding. 相似文献
11.
Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior 总被引:21,自引:0,他引:21
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Sander EE ten Klooster JP van Delft S van der Kammen RA Collard JG 《The Journal of cell biology》1999,147(5):1009-1022
Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts. 相似文献
12.
Chemoattractant-stimulated phagocytes increase their glucose uptake and divert energy production from glycolysis to the pentose phosphate pathway to generate NADPH. NADPH is a required cofactor for the NADPH oxidase to produce reactive oxygen metabolites, an important microbicidal tool in host defense. p21-Activated kinases (Paks) are regulated by the GTPases Rac and Cdc42 and control actin dynamics and phosphorylation of the oxidase component p47(phox). Here we report the interaction of Pak with phosphoglycerate mutase (PGAM)-B, an enzyme of the glycolytic pathway. Activated Pak1 inhibits glycolysis by association of its catalytic domain with PGAM-B and subsequent phosphorylation of the enzyme on serine residues 23 and 118, thereby abolishing PGAM activity. Leukocyte activation through chemoattractant receptors leads to Pak activation and transient inhibition of endogenous PGAM-B activity. Consistent with these observations, treatment of neutrophils with phosphoglycolic acid, a competitive PGAM-B inhibitor, increases upstream intermediates, thereby amplifying the respiratory burst. These results demonstrate that Rho GTPases regulate the glycolytic pathway through Pak and suggest a link between chemoattractant signaling and metabolic responses to enhance host defense. 相似文献
13.
Schlegel N Meir M Spindler V Germer CT Waschke J 《Journal of cellular physiology》2011,226(5):1196-1203
Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions. 相似文献
14.
Finkielstein CV Overduin M Capelluto DG 《The Journal of biological chemistry》2006,281(37):27317-27326
The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting. 相似文献
15.
Qian Y Liu KJ Chen Y Flynn DC Castranova V Shi X 《The Journal of biological chemistry》2005,280(5):3875-3884
Although arsenic is a human carcinogen, the molecular mechanisms of its action remain to be understood. The present study reports that exposure to arsenic induced actin filament reorganization, resulting in lamellipodia and filopodia structures through the activation of Cdc42 in SVEC4-10 endothelial cells. It was also found that arsenic induced the formation of the superoxide anion (O2*) in SVEC4-10 cells. Immunoprecipitation and Western blotting analysis demonstrated that arsenic stimulation induced serine phosphorylation of p47phox, a key component of NADPH oxidase, indicating that arsenic induces O2* formation through NADPH oxidase activation. Inhibition of arsenic-induced actin filament reorganization by either overexpression of a dominant negative Cdc42 or pretreatment of an actin filament stabilizing regent, jasplakinolide, abrogated arsenic-induced NADPH oxidase activation, showing that the activation of NADPH oxidase was regulated by Cdc42-mediated actin filament reorganization. This study also showed that overexpression of a dominant negative Rac1 was sufficient to abolish arsenic-induced O2*- production, implying that Rac1 activities are required for Cdc42-mediated NADPH oxidase activation in response to arsenic stimulation. Furthermore, arsenic stimulation induced cell migration, which can be inhibited by the inactivation of either Cdc42 or NADPH oxidase. Taken together, the results indicate that arsenic is able to activate NADPH oxidase through Cdc42-mediated actin filament reorganization, leading to the induction of an increase in cell migration in SVEC4-10 endothelial cells. 相似文献
16.
NADPH oxidases: not just for leukocytes anymore! 总被引:23,自引:0,他引:23
In addition to their role in bacterial killing by leukocytes, reactive oxygen species (ROS) have been increasingly recognized as important components of signaling and host defense in other cell types. The formation of ROS in both phagocytic- and non-phagocytic cells involves membrane-localized NADPH oxidases (Noxs). Nox proteins show structural homology to the cytochrome b(558) of leukocytes but, until recently, their regulation has been poorly understood. Here, we describe our current understanding of Nox function, and discuss emerging paradigms for regulation of Nox activity by Rac GTPase and/or other cytosolic components. 相似文献
17.
SPECs, small binding proteins for Cdc42 总被引:1,自引:0,他引:1
Pirone DM Fukuhara S Gutkind JS Burbelo PD 《The Journal of biological chemistry》2000,275(30):22650-22656
The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling. We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities. The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain. Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA. Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain. Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity. Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain. These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities. 相似文献
18.
PTP mu is expressed in the developing nervous system and promotes growth and guidance of chick retinal ganglion cells. Using a newly developed growth cone rearrangement assay, we examined whether the small G-proteins were involved in PTP mu-dependent signaling. The stimulation of retinal cultures with purified PTP mu resulted in a striking morphological change in the growth cone, which becomes dominated by filopodia within 5 min of addition. This rearrangement in response to PTP mu stimulation was mediated by homophilic binding. We perturbed GTPase signaling using Toxin B, which inhibits Cdc42, Rac, and Rho, as well as the toxin Exoenzyme C3 that inhibits Rho. The PTP mu-induced growth cone rearrangement was blocked by Toxin B, but not by Exoenzyme C3. This result suggests that either Cdc42 or Rac are required but not Rho. To determine which GTPase was involved in PTP mu signaling, we utilized dominant-negative mutants of Cdc42 and Rac. Dominant-negative Cdc42 blocked PTP mu-induced rearrangement, while wild-type Cdc42 and dominant-negative Rac did not. Together, these results suggest a molecular signaling cascade beginning with PTP mu homophilic binding at the plasma membrane and the activation of Cdc42, which acts on the actin cytoskeleton to result in rearrangement of the growth cone. 相似文献
19.
《Cellular signalling》2014,26(9):1975-1984
Cytoskeletal reorganization is crucial for platelet adhesion and thrombus formation to avoid excessive bleeding. Major regulators of cytoskeletal dynamics are small GTPases of the Rho family. Rho GTPases become activated by G-protein coupled receptor activation, downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors and by outside-in signaling of integrins. They act as molecular switches and cycle between active and inactive states. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP to terminate Rho signaling. Nadrin is a RhoGAP that was recently identified in platelets. Five Nadrin isoforms are known consisting of a unique GAP and an N-terminal BAR domain responsible for the selective regulation of RhoA, Cdc42 and Rac1. Besides BAR domain mediated regulation of Nadrin GAP activity nothing is known about the regulation of Nadrin and the impact on cytoskeletal reorganization. Here we show that Nadrin becomes tyrosine phosphorylated upon platelet activation. We found Src family proteins (Src, Lyn, Fyn) to be responsible to control Nadrin GAP activity by phosphorylation. Interestingly, phosphorylation of Nadrin leads to tightly regulated Rho activation that was found to be Nadrin isoform- and (Rho) target-specific. Src-phosphorylation of Nadrin5 mediated inactivation of Cdc42 while RhoA and Rac1 became activated upon Src-mediated phosphorylation of Nadrin2. Our results suggest a critical role for spatial and temporal regulation of Nadrin and thus for the control of Rho GTPases in platelets. 相似文献
20.
Neutrophils act as the first line of innate immune defense against invading microorganisms during infection and inflammation. The tightly regulated production of reactive oxygen species (ROS) through activation of NADPH oxidase is a major weapon used by neutrophils and other phagocytic leukocytes to combat such pathogens. Cellular adhesion signals play important physiological roles in regulating the activation of NADPH oxidase and subsequent ROS formation. We previously showed that the initial suppression of the oxidase response of chemoattractant-stimulated adherent neutrophils is mediated via inhibition of Vav1-induced activation of the NADPH oxidase regulatory GTPase Rac2 by adhesion signals. In this study we show that prior exposure of neutrophils to a number of cytokines and inflammatory mediators, including TNF-alpha, GM-CSF, and platelet-activating factor, overcomes the adhesion-mediated suppression of ROS formation. Proline-rich tyrosine kinase 2 (pyk2) activity is enhanced under these conditions, correlating with the restoration of Vav1 and Rac2 activities. Both dominant negative pyk2 and a pyk2-selective inhibitor prevented restoration of ROS production induced by TNF-alpha, GM-CSF, and platelet-activating factor, and this loss of pyk2 activity resulted in decreased Vav1 tyrosine phosphorylation and subsequent Rac2 activation. Our studies identify pyk2 as a critical regulatory component and a molecular switch to overcome the suppression of leukocyte oxidant generation by cell adhesion. This activity constitutes a mechanism by which cytokines might lead to rapid elimination of invading pathogens by adherent neutrophils under normal conditions or enhance tissue damage in pathological states. 相似文献