Molecular Cloning of Two Types of GAP Complementary DNA from Human Placenta

Trahey, Meg; Wong, Gina; Halenbeck, Robert; Rubinfeld, Bonnee; Martin, George A.; Ladner, Martha; Long, Christopher M.; Crosier, Walter J.; Watt, Ken; Koths, Kirston; McCormick, Frank

doi:10.1126/science.3201259

Cited by 480 publications

(227 citation statements)

References 10 publications

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“…rasGAP is a GTPase activating protein and it functions as a negative regulator of Ras signaling by stimulating the intrinsic rate of Ras GTPase activity (Trahey et al, 1988). Deletion of the rasGAP gene (Henkemeyer et al, 1995) produces a very pleiotropic phenotype as would be expected for a key enzyme which rests downstream of multiple signaling pathways.…”

Section: Discussionmentioning

confidence: 99%

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

1998

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Tek/Tie2 is an endothelial cell-speci®c receptor tyrosine kinase that has been shown to play a role in vascular development of the mouse. Targeted mutagenesis of both Tek and its agonistic ligand, Angiopoietin-1, result in embryonic lethality, demonstrating that the signal transduction pathway(s) mediated by this receptor are crucial for normal embryonic development. In an attempt to identify downstream signaling partners of the Tek receptor, we have used the yeast two-hybrid system to identify phosphotyrosine-dependent interactions. Using this approach, we have identi®ed a novel docking molecule called Dok-R, which has sequence and structural homology to p62 dok and IRS-3. Mapping of the phosphotyrosine-interaction domain within Dok-R shows that Dok-R interacts with Tek through a PTB domain. Dok-R is coexpressed with Tek in a number of endothelial cell lines. We show that coexpression of Dok-R with activated Tek results in tyrosine phosphorylation of Dok-R and that rasGAP and Nck coimmunoprecipitate with phosphorylated Dok-R. Furthermore, Dok-R is constitutively bound to Crk presumably through the proline rich tail of Dok-R. The cloning of Dok-R represents the ®rst downstream substrate of the activated Tek receptor, and suggests that Tek can signal through a multitude of pathways.

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Section: Discussionmentioning

confidence: 99%

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

1998

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“…The 1433 bp EcoRV fragment of human GAP (nucleotides 2123 ± 3556) from clone 101, kindly provided by Frank McCormick, UCSF (Trahey et al, 1988) was cloned into the unique PvuII site downstream of the GAL1 promoter in the pYES2 plasmid (Invitrogen). This pYES2-GAP plasmid was modi®ed to code for ADE2 instead of URA3 by ligating the 7.0 kbp blunted fragment of pYES2-GAP plasmid digested with NdeI and ApaI to a 4.5 kbp blunted fragment encoding ADE2 originating from the pM1 plasmid (Segal et al, 1993), digested with BglI and EcoRI.…”

Section: Construction Of the Gap Inducible Allelementioning

confidence: 99%

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements

et al. 2001

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We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA-and KBRas) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modi®ed Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar e ciencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting di erent structural requirements for GRP. Analysis of Ras-mediated gene activation in murine ®broblasts con®rmed these results. Oncogene (2001) 20, 2091 ± 2100.

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“…Guanine nucleotide exchange factors catalyze the dissociation of GDP from Ras and thus promote the loading of GTP to regenerate the active state. The accessory GTPase-activating proteins (GAPs) negatively regulate the GTP-bound state by increasing the slow intrinsic hydrolysis rate of Ras by factors of up to 10 5 (8)(9)(10)(11)(12). Oncogenic mutants of Ras are found in 25-30% of human tumors (3,13,14).…”

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confidence: 99%

The arginine finger of RasGAP helps Gln-61 align the nucleophilic water in GAP-stimulated hydrolysis of GTP

Resat

Straatsma

Dixon

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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The Ras family of GTPases is a collection of molecular switches that link receptors on the plasma membrane to signaling pathways that regulate cell proliferation and differentiation. The accessory GTPase-activating proteins (GAPs) negatively regulate the cell signaling by increasing the slow intrinsic GTP to GDP hydrolysis rate of Ras. Mutants of Ras are found in 25-30% of human tumors. The most dramatic property of these mutants is their insensitivity to the negative regulatory action of GAPs. All known oncogenic mutants of Ras map to a small subset of amino acids. Gln-61 is particularly important because virtually all mutations of this residue eliminate sensitivity to GAPs. Despite its obvious importance for carcinogenesis, the role of Gln-61 in the GAP-stimulated GTPase activity of Ras has remained a mystery. Our molecular dynamics simulations of the p21ras-p120GAP-GTP complex suggest that the local structure around the catalytic region can be different from that revealed by the x-ray crystal structure. We find that the carbonyl oxygen on the backbone of the arginine finger supplied in trans by p120GAP (Arg-789) interacts with a water molecule in the active site that is forming a bridge between the NH 2 group of the Gln-61 and the ␥-phosphate of GTP. Thus, Arg-789 may play a dual role in generating the nucleophile as well as stabilizing the transition state for POO bond cleavage.L ow molecular weight GTP-binding proteins, like p21Ras, act as molecular switches in cellular signaling pathways that control cell proliferation and differentiation (for extensive reviews see refs. 1-7). In the GTP-bound on (active) state, Ras interacts with effector molecules and transmits signals to the next downstream component. The hydrolysis of GTP to GDP switches Ras to the off (inactive) state. Guanine nucleotide exchange factors catalyze the dissociation of GDP from Ras and thus promote the loading of GTP to regenerate the active state. The accessory GTPase-activating proteins (GAPs) negatively regulate the GTP-bound state by increasing the slow intrinsic hydrolysis rate of Ras by factors of up to 10 5 (8-12). Oncogenic mutants of Ras are found in 25-30% of human tumors (3,13,14). Mutations of Gln-61 increase the activation barrier for Ras hydrolysis of GTP by 1.5-2 kcal͞mol, thereby decreasing the rate by at least one order of magnitude depending on the mutant (15, 16). The most dramatic property of these mutants is their insensitivity to the negative regulatory action of GAPs. Mutations of Gln-61 have been found that reduce the rate of GAP-stimulated hydrolysis by as much as 10 6 -fold (17, 18), which corresponds to an increase in the activation barrier by 8.5 kcal͞mol. There is little doubt that the oncogenic properties of Gln-61 mutations are caused by their impact on the intrinsic and GAP-stimulated rates of GTP hydrolysis; however, the biochemical origins of these effects are still not entirely clear.The proton-transfer step before nucleophilic attack on the ␥-phosphate of GTP is a critical point in understanding the in...

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Molecular Cloning of Two Types of GAP Complementary DNA from Human Placenta

Cited by 480 publications

References 10 publications

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements

The arginine finger of RasGAP helps Gln-61 align the nucleophilic water in GAP-stimulated hydrolysis of GTP

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