Browsing by Author "Wittinghofer, Alfred"

Now showing 1 - 6 of 6
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    Biological properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction
    (1988-08) John, Jasson; Frech, Matthias; Wittinghofer, Alfred
    Kinetic studies performed on p21H guanine nucleotide complexes with and without Mg2+ show that point mutations at positions 12, 59, and 61 each have a different effect on the rate of nucleotide dissociation. Double mutants with a combination of these amino acid substitutions reveal that the effects of each mutation on these kinetics are interactive (nonadditive) for positions 12 and 59 and approximately additive for the positions 12 and 61. The magnitude and direction of the effects seen are dependent on the nature of the nucleotide and whether or not the complexes contain Mg2+. All the mutants have reduced GTPase activity. It is also shown that the autophosphorylation reaction velocity is of first order with respect to the protein concentration and that this reaction is an intramolecular one, which takes place as a side reaction of the GTPase reaction. The autophosphorylation is not reversible under the experimental conditions. The covalently bound phosphate does not decrease the nucleotide-binding ability of the protein nor does it change the relative affinity of the protein for GTP versus GDP. The results are discussed in terms of the structural model and function of p21H.
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    Borasio, G.D. et al. ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons. Neuron 2, 1087-1096
    (1989-01) Borasio, Gian D.; John, Jasson; Wittinghofer, Alfred; Barde, Y. A.; Sendtner, Michael; Heumann, Rolf
    Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.
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    Characterisation of the metal-ion - GDP complex at the active sites of transforming and nontransforming p21 proteins by observation of the 17O-Mn superhyperfine coupling and by kinetic methods
    (1987-01) Feuerstein, Juergen W.; Kalbitzer, Hans R.; John, Jasson; Goody, Roger S.; Wittinghofer, Alfred
    Kinetic studies on the interaction of three Ha-ras-encoded p21 proteins with GDP and MgGDP have yielded values for the association (10(6)-10(7) M-1 s-1) and dissociation (10(-3)-10(-5) s-1) rate constants at 0 degrees C. Dramatic differences in the rate constants were not observed for the three proteins. Under non-physiological conditions (absence of Mg2+), the rate constant for GDP release was an order of magnitude faster for the viral protein p21v than for the cellular form p21c or the T24 mutant p21t, but this was reduced to a factor of about 3 in the presence of Mg2+. In all cases, there was an increase of about one order of magnitude in the rate of GDP release on removing magnesium. The binding affinities ranged from 5.7 X 10(10) M-1 for p21c to 1.3 X 10(11) M-1 for p21v. Electron paramagnetic resonance (EPR) measurements on Mn2+ bound together with stereospecifically 17O-labelled GDP showed direct coordination of a beta-phosphate oxygen to the metal ion with a superhyperfine coupling constant of 0.16-0.22 mT, but no interaction with the alpha-phosphate oxygens at the active site of all three proteins. The association constant of Mn(II) to p21 proteins in the absence of nucleotides was estimated to be greater than 10(5) M-1. In agreement with the EPR results, experiments on the metal ion dependence of the binding of thiophosphate analogs of GDP provided further evidence for the absence of direct coordination of the metal ion to the alpha-phosphate group. These results have been used to construct a model for the interactions of Mg X GDP with the active site of p21 proteins.
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    Crystallization and preliminary X-ray analysis of the human c-H-ras-oncogene product p21 complexed with GTP analogues
    (Elsevier, 1989-03) Scherer, Anna; John, Jasson; Linke, Rosita; Goody, Roger S.; Wittinghofer, Alfred; Pai, Emil F.; Homes, K. C.
    The catalytic domain (amino acid residues 1 to 166) of the human ras-oncogene product p21 complexed with the GTP analogues beta,gamma-imido-GTP (GMPPNP), beta,gamma-methylene-GTP (GMPPCP), and guanosine-5'-(gamma-thiotriphosphate) (GTP gamma S) have been been crystallized. Crystals of the GMPPNP and GMPPCP complexes are well suited for high resolution X-ray crystallography. They belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit cell axes a=b=40.3 A and c = 162.2 A.
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    ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons
    (Elsevier, 2009-01) Borasio, Gian D.; John, Jasson; Wittinghofer, Alfred; Barde, Y. A.; Sendtner, Michael; Heumann, Rolf
    Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal rootganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-an-choring, palmityl-accepting cysteine. These results sug-gest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.
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    Tucker, J. et al. Expression of p21 proteins in Escherichia coli and stereochemistry of th nucleotide-binding site. EMBO J. 5, 1351-1358
    (1986-06) Tucker, J.; Sczakiel, Georg; Feuerstein, Juergen W.; John, Jasson; Goody, Roger S.; Wittinghofer, Alfred
    v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.