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Results for “RAC2 GTP-Binding Protein”

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PubMed1999

Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense.

In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions.

Actins↗

PubMed1993

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↗

PubMed1997

Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of the responsiveness to guanine nucleotide dissociation stimulator.

The Rac GTPases are currently being subjected to intensive study due to their involvement in a wide array of cellular phenomena. Many studies of Rac function have relied upon the use of relatively uncharacterized Rac dominant active, dominant negative, and effector domain mutants on the basis of the analogy to Ras structure. We have generated and purified such Rac2 mutants and characterized their guanine nucleotide binding properties in vitro. The Rac2 G12V and Q61L activating mutations were shown to hydrolyze bound GTP very slowly and were unresponsive to p190 Rac GTPase-activating protein. Distinct differences in the kinetics of nucleotide binding to individual mutant proteins were observed, accounting for the behavior of these proteins in biological assays. The structural features required for the responsiveness of Rac2 to the guanine nucleotide exchange protein smgGDS were examined. We show that guanine nucleotide exchange by smgGDS is dependent upon intact switch 1 and switch 2 regions in Rac2. Functional interactions between the switch 1 and switch 2 regions and the G12V mutation of Rac2 are described. These data form the basis for rational use of Rac mutants in biological studies.

GTP Phosphohydrolases↗

PubMed1996

Membrane association of Rac is required for high activity of the respiratory burst oxidase.

NADPH-dependent superoxide generation can be reconstituted in a cell-free system using recombinant cytosolic factors (p47-phox, p67-phox, and Rac) plus flavocytochrome b558. Rac1 and Rac2 are closely related small GTPases, differing primarily in the C-terminal 10 residues where Rac1 but not Rac2 contains a polybasic sequence. In their nonisoprenylated forms, Rac1 was highly effective in reconstituting NADPH oxidase activity (low EC50, high Vmax), whereas Rac2 was only minimally effective (high EC50, low Vmax). In contrast, low concentrations of isoprenylated Rac1 and Rac2 both supported high rates of superoxide generation. Like full length Rac2, truncated forms of both Rac1 and Rac2 in which the C-terminal 10 residues were eliminated were poorly activating, pointing to the C terminus of Rac1 as a determinant of activity. Mutation of single positively charged residues in the C terminus of nonisoprenylated Rac1 markedly reduced its ability to support superoxide generation, affecting both its EC50 and the Vmax. In contrast, mutation or truncation of the C terminus failed to affect the activation of PAK, a Rac-regulated protein kinase. The EC50 for Rac1 increased with increasing salt concentrations, whereas that of Rac2 was independent of salt, implicating the involvement of electrostatic forces for the former. Using flavocytochrome b558 reconstituted into phosphatidylcholine vesicles, the EC50 for Rac1 but not Rac2 decreased (increased binding) when an acidic phospholipid (phosphatidylinositol) was present, supporting a role for the Rac1 polybasic C terminus in binding to the membrane. A model in which Rac must associate simultaneously both with p67-phox and with the membrane to activate the NADPH oxidase can account for the above observations.

Amino Acid Sequence↗

PubMed1997

Enhanced apoptosis in the thymus of transgenic mice expressing constitutively activated forms of human Rac2GTPase.

Rac proteins constitute a subgroup of the Rho family of small GTPases and include Rac1, which is expressed ubiquitously, and Rac2, a highly homologous protein only expressed in myelo-monocytic and lymphoid cell lineages. In fibroblasts, Rac1 plays a crucial role in control of actin cytoskeleton organisation, cell growth and Ras-induced transformation. In phagocytes, Rac1 and Rac2 regulate a specific enzymatic complex, NADPH oxidase. These multiple functions have been ascribed to Rac proteins only on the basis of cell culture and in vitro biochemical studies. To examine the role of Rac2 in vivo in a T cell lineage, we have expressed either wild-type or constitutively-activated forms of human Rac2 (Rac2V12 and Rac2L61) in transgenic mice under control of the thymus specific lck proximal promoter. We report here a striking atrophy of the thymus in mice expressing even low levels of either of the activated mutants of Rac2, while expression of Rac2wt has no effect. This phenotype is correlated with a marked decrease in the number of double positive (CD4+ CD8+) and single positive (CD4+ CD8- and CD8+ CD4-) thymocytes. Cellular and molecular analyses demonstrate that this defect is due to an increase in apoptosis among thymocytes. As Rac2 is normally expressed in thymocytes and activated T cells, we propose that Rac2 dependent pathways could play an important role in control of growth and death of T cells.

Animals↗

PubMed1997

RhoGDIgamma: a GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas.

GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activation of GTPases and may also be critical for the cellular compartmentalization of GTPases. RhoGDI and GDI/D4 are two currently known GDIs for the Rho-subfamily of GTPases. Using their cDNAs to screen a human brain cDNA library under low stringency, we have cloned a homologous cDNA preferentially expressed at high levels in brain and pancreas. The predicted protein, named RhoGDIgamma, is approximately 50% identical to GDI/D4 and RhoGDI. It binds to CDC42 and RhoA with less affinity compared with RhoGDI and does not bind with Rac1, Rac2, or Ras. RhoGDIgamma functions as a GDI for CDC42 but with approximately 20 times less efficiency than RhoGDI. Immunohistochemical studies showed a diffuse punctate distribution of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. Overexpression of the protein in baby hamster kidney cells caused the cells to round up with loss of stress fibers. A distinct hydrophobic amino terminus in RhoGDIgamma, not seen in the other two RhoGDIs, could provide a mechanism for localization of the GDI to specific membranous compartment thus determining function distinct from RhoGDI or GDI/D4. Our results provide evidence that there is a family of GDIs for the Rho-related GTPases and that they differ in binding affinity, target specificity, and tissue expression. We propose that RhoGDI be renamed RhoGDIalpha and GDID4 be renamed RhoGDIbeta. The new GDI should widen the scope of investigation of this important class of regulatory protein.

Amino Acid Sequence↗

PubMed1996

Cleavage arrest of early frog embryos by the G protein-activated protein kinase PAK I.

PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.

Animals↗

PubMed1997

Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase.

Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67(phox) but not p47(phox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67(phox) with a 1:1 stoichiometry and with Kd values of 120 and 60 nM, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801) previously identified two regions in Rac1 that are important for activity: the "effector region" (residues 26-45) and the "insert region" (residues 124-135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b558.

Animals↗

PubMed1998

Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPH oxidase component p67(phox).

Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67(phox), but not with the other oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore, E., Ahmed, S., Best, A., Kozma, R. , Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67(phox) has been characterized further. Rac1 and Rac2 can bind to p67(phox) amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling. Deletion of p67(phox) C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by approximately 8-fold. p21(Cdc42Hs/Rac)-activated kinase (PAK) phosphorylates p67(phox) amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67(phox) in control of the NADPH oxidase.

3T3 Cells↗

PubMed1998

Structural requirements for PAK activation by Rac GTPases.

The Rho family GTPases, Rac1 and Rac2, regulate a variety of cellular functions including cytoskeletal reorganization, the generation of reactive oxygen species, G1 cell cycle progression and, in concert with Ras, oncogenic transformation. Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase p21-activated kinase (PAK) is a prime candidate for mediating some of Rac's cellular effects. This report shows that Rac1 binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structural elements on Rac and PAK that are important for optimal binding and activation. The most notable difference between the highly homologous Rac isomers is the composition of their C-terminal polybasic domains. Mutation of these six basic residues in Rac1 to neutral amino acids dramatically decreased the ability of Rac1 to bind PAK1 and almost completely abolished its ability to stimulate PAK activity. Moreover, replacing the highly charged polybasic domain of Rac1 with the less charged domain of Rac2 (and vice versa) completely reversed the PAK binding/activation properties of the two Rac isomers. Thus, polybasic domain differences account for the disparate abilities of Rac1 and Rac2 to activate PAK. PAK proteins also contain a basic region, consisting of three contiguous lysine residues (Lys66-Lys67-Lys68), which lies outside of the previously identified Cdc42/Rac-binding domain. Mutation of these Lys residues to neutral residues decreased PAK binding to activated Rac1 and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rac1, Rac2, and Cdc42 proteins in vivo. In contrast, mutation of lysines 66-68 to basic Arg residues did not decrease (and in some cases enhanced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effector domain that may allow the two Rac isomers to activate different effector proteins. In addition, our results indicate that a basic region in PAK is required for PAK activation and that binding of Rac/Cdc42 to PAK is not sufficient for kinase activation.

Amino Acid Sequence↗

PubMed1998

Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation.

The Rho family GTPases are tightly regulated between the active GTP-bound state and the inactive GDP-bound state in a variety of signal transduction processes. Here the Rho family members Cdc42, Rac2, and RhoA were found to form reversible homodimers in both the GTP- and the GDP-bound states. The homophilic interaction of Cdc42 and Rac2, but not RhoA, in the GTP-bound state, caused a significant stimulation of the intrinsic GTPase activity, i.e. the activated form of Cdc42 and Rac2 acts as GTPase-activating proteins toward Cdc42-GTP or Rac2-GTP. The dimerization of the GTPases appeared to be mediated by the carboxyl-terminal polybasic domain, and the specific GTPase-activating effects of Cdc42 and Rac2 were also attributed to the structural determinant(s) in the same region of the molecules. Moreover, similar to the case of Cdc42 and Cdc42GAP interaction, Cdc42-GDP interacted with tetrafluoroaluminate and Cdc42-GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) to form a transition state complex of the GTPase-activating reaction in which the carboxyl-terminal determinant(s) of the GTPgammaS-bound Cdc42 plays a critical role. These results provide a rationale for the fast rate of intrinsic GTP hydrolysis by Cdc42 and Rac and suggest that dimerization may play a role in the negative regulation of specific Rho family GTPases mediated by the carboxyl-terminal polybasic domain.

Aluminum Compounds↗

PubMed1999

Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.

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.

Animals↗

PubMed1999

Identification and functional characterization of the murine Rac2 gene promoter.

Rac2, a member of the Rho family of GTPases, is highly expressed in myeloid cells and is a regulator of the NADPH-oxidase complex. A murine genomic clone was isolated that contains the 5' end and putative promoter region of the Rac2 gene. Ribonuclease protection experiments detected 13 transcription initiation sites scattered 50 to 130 bp upstream of the translation initiation site. Transient transfection studies revealed that -7 kb to +31 bp (relative to the strongest transcription initiation site) of the Rac2 gene 5'-flanking region exhibited strong promoter activity in both RAW 264.7 macrophage cells that express the endogenous Rac2 gene and NIH-3T3 fibroblast cells that do not express the endogenous gene. Truncated Rac2 promoter fragments containing as little as the -74 to +31 bp sequence produced full transcriptional activity. However, a -57 to +31 promoter fragment directed significantly less transcription, and a -39 to +31 promoter fragment was transcriptionally inactive. In vitro binding assays revealed sequence-specific and widely expressed DNA-binding activities that interacted within the -74 to -58 Rac2 promoter cis element. Oligonucleotide competition and antibody disruption studies indicated that these complexes contained the transcription factors Spl and Sp3. Specific ablation of the Sp1/Sp3 binding site significantly decreased Rac2 promoter activity in both RAW 264.7 and NIH-3T3 cells. Additional cis elements may be required to restrict Rac2 promoter activity to hematopoietic cells expressing the endogenous gene.

3T3 Cells↗

PubMed1998

Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42.

We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and guanosine triphosphate (GTP)gammaS over about 20-30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPgammaS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca2+ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca2+ and GTPgammaS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for GE, GTPases that mediate exocytosis in cells of hematopoeitic origin.

Bacterial Proteins↗

PubMed1998

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.

Animals↗

PubMed1997

Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells.

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.

Cladosporium↗

PubMed1993

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↗

PubMed1995

Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation.

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47-phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.

Animals↗