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Query: UNIPROT:P06889 (Mol)
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The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze Gp4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.
Mol Cell Biochem 1992 May 13
PMID:Biochemical characterization of Artemia ras p21. 151 32

Proteins of the ras family of oncogenes have been implicated in signal transduction pathways initiated by protein kinase C (PKC) and by tyrosine kinase oncogenes and receptors, but the role that ras plays in these diverse signalling systems is poorly defined. The activity of ras proteins has been shown to be controlled in part by a cellular protein, GAP (GTPase-activating protein), that negatively regulates p21c-ras by enhancing its intrinsic GTPase activity. Thus, overexpression of GAP provides a tool for determining the step(s) in signal transduction dependent on p21c-ras activity. In this paper, we report that overexpression of GAP blocks the phorbol ester (tetradecanoyl phorbol acetate [TPA])-induced activation of p42 mitogen-activated protein kinase (p42mapk), c-fos expression, and DNA synthesis. GAP overexpression did not block responses to serum or fluoroaluminate. Moreover, not all biochemical events elicited by TPA were affected by GAP overexpression, as increased glucose uptake and phosphorylation of MARCKS, a major PKC substrate, occurred normally. Reduction of GAP expression to near normal levels restored the ability of the cells to activate p42mapk in response to TPA. These findings suggest that ras and GAP together play a key role in a PKC-dependent signal transduction pathway which leads to p42mapk activation and cell proliferation.
Mol Cell Biol 1992 Mar
PMID:Regulation of tetradecanoyl phorbol acetate-induced responses in NIH 3T3 cells by GAP, the GTPase-activating protein associated with p21c-ras. 154 25

Common practice emphasizes significant sequence similarities between different members of protein families. These similarities presumably reflect on evolutionary conservation of structurally and functionally essential residues. The nonconserved regions, on the other hand, may be either selectively neutral or differentiated. We propose several distributional sequence statistics (e.g., clustering of charged residues, compositional biases, and repetitive patterns) as indicators of differentiation events. These ideas are illustrated with various examples, including comparisons among G protein-coupled receptors, herpesvirus proteins, and GTPase-activating proteins.
Mol Biol Evol 1992 Jan
PMID:Significant similarity and dissimilarity in homologous proteins. 155 37

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-1. Mutations of residues in loop L1 (Gly-12 and Gly-13), in loop L2 (Thr-35 and Asp-38), and in loop L4 (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.
Mol Cell Biol 1992 May
PMID:Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity. 156 40

Mammalian sperm possess a guanine nucleotide-binding regulatory protein (G protein), with properties similar to Gi, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)-mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates Gi proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high-affinity GTPase activity and GTP gamma [35S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm-associated Gi protein. An increase in GTP hydrolysis (approximately 50% over basal activity) and GTP gamma [35S] binding (approximately 25-60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [32P]ADP-ribosylation of a Mr = 41,000 sperm Gi protein, suggesting that the increase in GTPase activity and GTP gamma [35S] binding is associated with the activation of a PT-sensitive sperm G protein(s). The ability of the ZP to stimulate high-affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface-associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.
Mol Reprod Dev 1992 Apr
PMID:Activation of a G protein in mouse sperm by the zona pellucida, an egg-associated extracellular matrix. 157 Nov 63

Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.
Mol Biol Cell 1992 May
PMID:Ras-related GTPases and the cytoskeleton. 161 Nov 53

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.
Mol Cell Biol 1992 Jul
PMID:Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells. 162 Jan 32

Members of the GTPase superfamily are extremely important in regulating membrane signalling pathways in all cells. This review focuses on membrane-associated GTPases that have been described in prokaryotes. In bacteria, LepA and NodQ are very similar to protein synthesis elongation factors but apparently have membrane-related functions. The amino acid sequences of FtsY and Ffh are clearly related to eukaryotic factors involved in protein secretion. Obg and Era are not closely related to any GTPase subgroup according to amino acid sequence comparisons, but they are essential for viability. In spite of similarities to well-studied eukaryotic proteins the signalling pathways of these cellular regulators, with the exception of NodQ, have not yet been elucidated.
Mol Microbiol 1992 May
PMID:Membrane-associated GTPases in bacteria. 164 Aug 28

The alpha subunit of the guanine nucleotide-binding regulatory protein GS mediates stimulation of adenylyl cyclase activity. This subunit, GS alpha, exists as two molecular weight forms, termed long and short, that differ by 14 or 15 amino acids. A physiological distinction between these two forms has yet to be defined. To compare the activities of these GS alpha isoforms, long and short forms of rat GS alpha were expressed in the cyc- variant of S49 murine lymphoma cells, which is deficient in endogenous GS alpha expression. By immunoblot analysis, the level of recombinant proteins in the clones expressing the long form of GS alpha was about twice that present in the clones expressing the short form of GS alpha or in the S49 wild-type cells. Both recombinant GS alpha proteins were sensitive to cholera toxin-catalyzed ADP-ribosylation, although the short form was labeled preferentially in both recombinant and S49 wild-type cell lines. In whole-cell assays, the clones expressing the long and short forms of GS alpha and the S49 wild-type cells gave comparable responses for stimulation of cAMP accumulation after challenge with (-)-isoproterenol, cholera toxin, or forskolin. In adenylyl cyclase assays with partially purified membranes, clones expressing the long form of GS alpha gave approximately twice the levels of cAMP in response to isoproterenol, guanosine-5'-O-(3-thio)triphosphate, NaF, or forskolin, compared with membranes from the clones expressing the short form of GS alpha or the S49 wild-type cells. However, when maximal adenylyl cyclase activity was normalized to the level of GS alpha protein in S49 wild-type cells, the cAMP productions were similar between all of the cell lines. In other membrane-based assays, the long and short forms of GS alpha were also equivalent in their dose response to isoproterenol and GTP, their kinetics of guanine nucleotide exchange and GTPase activity, and the induced high and low affinity states of the beta-adrenergic receptor in response to isoproterenol. In the latter radioligand binding analysis, membranes from the two clones expressing the long form of GS alpha consistently gave a greater proportion of the agonist high affinity state; however, this variation likely reflects the greater expression levels of GS alpha in these membranes. Thus, we conclude that the long and short forms of GS alpha expressed in S49 cyc- cells are very similar in their ability to stimulate adenylyl cyclase activity and to couple to beta-adrenergic receptors.
Mol Pharmacol 1991 Jun
PMID:Expression and characterization of the long and short splice variants of GS alpha in S49 cyc- cells. 164 45

A high-copy-number plasmid genomic library was screened for genes that when overexpressed down-regulate Ras protein activity in Saccharomyces cerevisiae. We report on the structure and characterization of one such gene, RPI1, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RPI1 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RPI1 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2Val-19 mutation, (ii) overexpressed RPI1 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2Val-19, and (iii) disruption of RPI1 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RPI1 appears to encode an inhibitor of Ras activity that shares a common feature with Ras GTPase-activating proteins in that it fails to down-regulate activated RAS2Val-19 function. We present evidence that the down-regulatory effect of RPI1 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.
Mol Cell Biol 1991 Aug
PMID:Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated ras function. 164 84


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