Purification of Rac2 from human neutrophils.
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Autoradiography↗
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Results for “RAC2 GTP-Binding Protein”
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Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation.
The respiratory burst oxidase is responsible for O2- production in stimulated neutrophils and B lymphocytes. Components of this oxidase include cytochrome b558, a membrane-bound flavohemoprotein; the cytosolic polypeptides p47phox and p67phox; and one or more small G proteins including Rac1, Rac2, and/or Rap1A. We found that when normal neutrophils were activated, small percentages of each of the cytosolic proteins p47phox, p67phox, and Rac2 were transferred to the membrane cytoskeleton. However, Rac2 was not transferred to the membrane during activation of p47phox-deficient neutrophils. In normal cells, some p47phox also became associated with the non-cytoskeletal portion of the plasma membrane, but p67phox, Rac2, and O(2-)-forming activity were restricted to the cytoskeleton. Neutrophil activation also causes the phosphorylation of multiple serines in p47phox. The most heavily phosphorylated forms of p47phox were found solely in the membrane cytoskeleton. These results suggest that 1) the membrane cytoskeleton participates in respiratory burst oxidase activation, 2) the fully phosphorylated p47phox is located in the active oxidase, which resides in the membrane cytoskeleton, and 3) Rac2 acts like a dedicated component of the respiratory burst oxidase.
Actins↗
Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox.
On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox, p47phox, p40phox, as well as the Ras-related G-proteins Rac1 and Rac2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b, forming a functional complex responsible for the production of oxygen radicals in phagocytes. In this paper we show that (a) in neutrophils from a patient with a form of chronic granulomatous disease (CGD) in which p67phox is absent, p47phox and Rac2, but not p40phox and Rac1 were translocated from the cytosol to the membrane on stimulation with formylmethionyl-leucylphenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA); (b) in neutrophils from a patient with a form of CGD in which p47phox is absent, p67phox, p40phox and Rac1 failed to associate with the membrane on stimulation with fMLP or PMA, whereas Rac2 was translocated as in normal neutrophils. We also show that in neutrophils from a patient lacking p67phox, the amount of cytosolic p40phox was decreased by about 40%. These findings indicate that, on neutrophil stimulation, p67phox mediates the translocation of p40phox and Rac1 from the cytosol to cell membranes and that Rac2 associates with the membranes independently of p47phox and p67phox.
Adult↗
Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavocytochrome b558.
When the neutrophil NADPH oxidase is activated to generate superoxide, the cytosolic components, p47phox, p67phox, and the GTP-binding protein Rac, become stably associated with the plasma membrane. The translocation of p47phox and p67phox is dependent on the presence in the membrane of the flavocytochrome b558, itself composed of two subunits, gp91phox and p22phox. In this study we have quantitated Rac1 and Rac2 in human neutrophils and show that > 96% of the Rac protein is Rac2 and that chronic granulomatous disease (CGD) neutrophils contain normal levels of the protein. We used a cell-free translocation system as well as intact normal and CGD neutrophils to determine whether the translocation of Rac2 is dependent upon the presence of the other oxidase components. When cell-free reactions contained any combination of normal, p47phox-deficient or p67phox-deficient cytosol and normal or flavocytochrome b558-deficient membranes, the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-dependent association of Rac2 with the re-isolated membranes was not significantly different from the control mixture of normal membranes and cytosol. In intact CGD neutrophils lacking p47phox or p67phox and stimulated with phorbol myristate acetate the translocation of Rac2 was also normal, and we conclude that Rac translocation can occur independently of the cytosolic NADPH oxidase components. In contrast, in the absence of flavocytochrome b558 from intact X-chromosome linked CGD neutrophils, Rac2 translocation was reduced to only 25% of the control value. On the basis of these observations we propose that while Rac2 can bind to a site distinct from either gp91phox or p22phox, it depends upon an interaction with the flavocytochrome b558 for maximal stability in the membrane.
Biological Transport↗
A dominant inhibitory version of the small GTP-binding protein Rac disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila.
The Rho subfamily of Ras-related small GTP-binding proteins is involved in regulation of the cytoskeleton. The cytoskeletal changes induced by two members of this subfamily, Rho and Rac, in response to growth factor stimulation, have dramatic effects on cell morphology. We are interested in using Drosophila as a system for studying how such effects participate in development. We have identified two Drosophila genes, DRacA and DRacB, encoding proteins with homology to mammalian Rac1 and Rac2. We have made transgenic flies bearing dominant inhibitory (N17DRacA), and wild-type versions of the DRacA cDNA under control of an Hsp70 promoter. Expression of the N17DRacA transgene during embryonic development causes a high frequency of defects in dorsal closure which are due to disruption of cell shape changes in the lateral epidermis. Embryonic expression of N17DRacA also affects germband retraction and head involution. The epidermal cell shape defects caused by expression of N17DRacA are accompanied by disruption of a localized accumulation of actin and myosin thought to be driving epidermal cell shape change. Thus the Rho subfamily may be generating localized changes in the cytoskeleton during Drosophila development in a similar fashion to that seen in mammalian and yeast cells. The Rho subfamily is likely to be participating in a wide range of developmental processes in Drosophila through its regulation of the cytoskeleton.
Actins↗
Purification and properties of Rac2 from human leukemia cells.
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Buffers↗
The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1.
NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b558 of cytosolic components p47phox, p67phox, and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67phox; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67phox. Finally, we observe that p67phox interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67phox and that Rac2 is the main p67phox-interacting GTPase in human cells.
Amino Acid Sequence↗
Differing structural requirements for GTPase-activating protein responsiveness and NADPH oxidase activation by Rac.
The Rac GTP-binding proteins regulate the actin cytoskeleton and the superoxide-forming NADPH oxidase of phagocytic leukocytes. These functions of Rac are determined by the GTP/GDP state of the protein, which can be modulated by GTPase-activating proteins (GAPs). The interaction of Ras with both downstream signaling targets and GAPs is mediated via an "effector" domain (amino acids 30-40). We demonstrate that the effector domain of Rac2 is required for both NADPH oxidase activation and actin assembly, but that mutations in this region do not decrease the responsiveness of Rac to GAPs. In contrast, mutations of residues 12 (Gly-->Val) or 61 (Gln-->Leu) inhibit both intrinsic- and GAP-stimulated GTP hydrolysis by Rac2. A double mutation in which both the effector domain and Q61L were modified restored NADPH oxidase activation and membrane ruffling, while the equivalent effector domain and G12V double mutation did not. The Rac2 Q61L mutant had an increased "affinity" for NADPH oxidase activation and for GAP binding as compared to the wild type or G12V proteins. These experiments suggest that Rac contains at least two "effector" interaction sites, and that changes in binding interactions at one of these sites may influence the function of the other.
Base Sequence↗
Spreading of differentiating human monocytes is associated with a major increase in membrane-bound CDC42.
In a search for a model cell system that might allow studies of the function of the Rho-related GTPase CDC42Hs in human cells, we measured the content and distribution of CDC42Hs in monocytes that were differentiating into macrophages. The total content of this protein increased 5- to 6-fold in phorbol ester-treated human monocytic THP-1 and U-937 cells and increased 13-fold in normal human blood monocytes. Moreover, membrane-associated CDC42Hs in these cells increased 13-fold and 30-fold, respectively, while cytosolic CDC42Hs increased only 3- and 6-fold. Measurements made specifically in U-937 cells showed that the increase in membrane CDC42Hs correlated closely with an increase in cell spreading. The changes in CDC42Hs in U-937 cells probably depended on increased mRNA translation and/or decreased protein degradation, since no change in CDC42Hs mRNA could be detected. Finally, the changes in CDC42Hs were relatively specific, since contents of the CDC42Hs-binding protein Rho-GDI and the Rho-related protein Rac2 were unaffected and no change in CDC42Hs occurred when the cells were stimulated by agonists that induce monocytes to differentiate into nonadherent cells. These findings show that marked changes in content and distribution of CDC42Hs occur when monocytes differentiate into macrophages, suggesting that membrane CDC42Hs may play a role in cell spreading.
Blotting, Western↗
Dissociation of Rac translocation from p47phox/p67phox movements in human neutrophils by tyrosine kinase inhibitors.
Cytosolic components of the phagocyte NADPH oxidase (p47phox, p67phox, and Rac2) translocate to the plasma membrane on cell activation where they interact with a membrane-bound cytochrome b to generate superoxide anion. Phosphorylation reactions are known to be important for activity of NADPH oxidase. Translocation of Rac2, p47phox, and p67phox were all enhanced in formyl-Met-Leu-Phe-stimulated neutrophils treated with 50 nM of the protein phosphatase 1/2A inhibitor calyculin A. Rac translocation was blocked by the tyrosine kinase inhibitors genistein (50 microM) and herbimycin (17 microM), whereas movement of p47phox and p67phox were not inhibited. Cell-free analysis of Rac translocation also demonstrated that translocation of p47phox and p67phox were not linked to the movement or availability of Rac2. Thus, Rac2 does not appear to regulate NADPH oxidase by controlling movements of the cytosolic components to the membrane-associated enzyme but may exert its effect at the level of the assembled complex. Tyrosine kinase activity is required for translocation of Rac in the chemoattractant-stimulated human neutrophil.
Alkaloids↗
Inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of small GTP-binding proteins. Lack of amino acid sequence specificity and importance of polybasic motif.
The small GTP-binding protein (G protein) Rac1 is an obligatory participant in the assembly of the superoxide (O2-.)-generating NADPH oxidase complex of macrophages. We investigated the effect of synthetic peptides, mapping within the near carboxyl-terminal domains of Rac1 and of related G proteins, on the activity of NADPH oxidase in a cell-free system consisting of solubilized guinea pig macrophage membrane, a cytosolic fraction enriched in p47phox and p67phox (or total cytosol), highly purified Rac1-GDP dissociation inhibitor for Rho (Rho GDI) complex, and the activating amphiphile, lithium dodecyl sulfate. Peptides Rac1-(178-188) and Rac1-(178-191), but not Rac2-(178-188), inhibited NADPH oxidase activity in a Rac1-dependent system when added prior to or simultaneously with the initiation of activation. However, undecapeptides corresponding to the near carboxyl-terminal domains of RhoA and RhoC and, most notably, a peptide containing the same amino acids as Rac1-(178-188), but in reversed orientation, were also inhibitory. Surprisingly, O2-. production in a Rac2-dependent cell-free system was inhibited by Rac1-(178-188) but not by Rac2-(178-188). Finally, basic polyamino acids containing lysine, histidine, or arginine, also inhibited NADPH oxidase activation. We conclude that inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of certain small G proteins is not amino acid sequence-specific but related to the presence of a polybasic motif. It has been proposed that such a motif serves as a plasma membrane targeting signal for a number of small G proteins (Hancock, J.F., Paterson, H., and Marshall, C.J. (1990) Cell 63, 133-139).
Amino Acid Sequence↗
Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.
The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.
Animals↗
Enhancement of protein kinase C-dependent O2 production in Epstein-Barr virus-transformed B lymphocytes by p120Ras-GAP antisense oligonucleotide.
The mammalian Ras GTPase-activating protein (p120Ras-GAP) interacts with activated members of the Ras superfamily of GTP-binding proteins to accelerate their deactivation by sharply increasing their rates of GTP hydrolysis. Among the Ras-family proteins interacting with p120Ras-GAP is Rap1A/Krev1, whose activity is not affected by p120Ras-GAP but which competes with Ras for p120Ras-GAP. A second protein that interacts with p120Ras-GAP is P190Rac-GAP, which activates the GTPase of guanine nucleotide-binding proteins of the Rho family (including Rac1 and Rac2). Both these p120Ras-GAP-binding proteins are of interest in connection with the regulation of the respiratory burst oxidase, Rap1A/Krev1 because it copurifies with cytochrome b558 and p190Ras-GAP because it inhibits the Rac2-dependent activation of the respiratory burst oxidase in a cell-free system. Using an 18-mer antisense oligonucleotide, we were able to decrease the expression of p120Ras-GAP in Epstein-Barr virus-transformed B lymphocytes. Under conditions where p120Ras-GAP expression was significantly depressed by antisense oligonucleotides, we observed a 40% increase in protein kinase C-dependent but not receptor-dependent O2 production. In contrast, sense and scrambled oligonucleotides had no effect on either p120Ras-GAP expression or O2 production. Our results suggest a role for p120Ras-GAP as a negative regulator in the protein kinase C-mediated activation of the respiratory burst oxidase.
B-Lymphocytes↗
Subcellular localization and kinetic characterization of guanine nucleotide binding proteins in normal rat and human pancreatic islets and transformed beta cells.
The subcellular localization and the kinetics of the GTPase activities of monomeric and heterotrimeric GTP-binding proteins were investigated in normal rat and human pancreatic islets and were compared to those obtained using a transformed hamster beta cell line (HIT cells). The [alpha-32P]GTP overlay technique revealed the presence of at least four low-molecular-mass proteins (approx. 20-27 kDa) in normal rat islets, which were enriched in the secretory granule fraction compared to the membrane fraction (with little abundance of these proteins in the cytosolic fraction). In contrast, in HIT cells, these proteins (at least six) were predominantly cytosolic. Three of these proteins were immunologically identified as rab3A, rac2, and CDC42Hs in islets as well as in HIT cells. In addition, pertussis toxin augmented the ribosylation of at least one heterotrimeric G-protein of about 39 kDa (probably G(i) and/or G(o)) in the membrane and secretory granule fractions of normal rat and human islets, whereas at least three such Ptx substrates (36-39 kDa) were found in HIT cell membranes. Kinetic activities revealed the presence of at least three such activities (Km for GTP of 372 nM, 2.2 microM, and 724 microM) in islet homogenates which were differentially distributed in various subcellular fractions; similar activities were also demonstrable in HIT cell homogenates. Thus, these studies demonstrate the presence of both monomeric G-proteins intrinsic to the secretory granules of normal rat islets which can be ascribed to beta cells; since these G-proteins are regulated by insulinotropic lipids (as described in the accompanying article), such proteins may couple the activation of phospholipases (endogenous to islets) to the exocytotic secretion of insulin. These findings also suggest that caution is necessary in extrapolating data concerning G-proteins from cultured, transformed beta cell lines to the physiology of normal islets, in view of both qualitative and quantitative differences between the two preparations.
Adult↗
Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages.
Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Characterization of rho GTPase family homologues in Drosophila melanogaster: overexpressing Rho1 in retinal cells causes a late developmental defect.
The rho family of GTPases has been implicated in regulating changes in cell morphology in response to extracellular signals. We have cloned three widely expressed members of this family from Drosophila melanogaster; a rho homologue (Rho1) and two rac homologues (Rac1 and Rac2). Flies harbouring a Rho1 transgene that is specifically expressed in the eye exhibit a dramatic dose dependent disruption of normal eye development. Flies bearing at least two copies of the transgene display a severe rough eye phenotype characterized by missing secondary and tertiary pigment cells, a substantial reduction in the number of photoreceptor cells and a grossly abnormal morphology of the rhabdomeres. Cell fate determination in the imaginal disc occurs normally and abnormalities become manifest late in pupariation, coincident with the phase when the cells undergo major morphological changes. This phenotype is modified by mutations at several other loci that have been implicated in signal transduction, but not by mutations in ras pathway components.
Amino Acid Sequence↗
Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins.
Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).
Amino Acid Sequence↗
A new member of the ras superfamily, the rac1 homologue from Caenorhabditis elegans. Cloning and sequence analysis of cDNA, pattern of developmental expression, and biochemical characterization of the protein.
A new member of the ras superfamily, designated CErac1 has been identified. The CErac1 cDNA clone was isolated from a Caenorhabditis elegans mixed stage library and encodes a protein of 191 amino acids with 82 and 79% identity to human rac1 and rac2 proteins, respectively. The CErac1 cDNA maps to a position on C. elegans chromosome IV in close proximity to cha-1, a choline acetyltransferase gene. The CErac1 cDNA hybridizes to two mRNAs (1.7 and 0.9 kilobases). Their expression is developmentally regulated, that of the more abundant 1.7 kilobases being highest at the embryonic stage and decreasing dramatically during development with 10% of the embryonic level in adult nematodes. The glutathione-S-transferase/CErac1 fusion protein expressed in Escherichia coli binds GTP and exhibits intrinsic GTPase activity. The GTPase activity of the CErac1 protein is stimulated by human n-chimaerin, a GTPase-activating protein for p21 rac1. These data suggest a role of CErac1 in C. elegans early development. The conserved biochemical properties indicate that further characterization of CErac1 by genetic analysis will be helpful in elucidating not only its role in the signal transduction, but also the biological function of its mammalian homologues.
Amino Acid Sequence↗