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Molecular and Cellular Biology, September 1999, p. 6297-6305, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Ras Mutant D119N Is Both Dominant Negative and Activated

Robbert H. Cool,* Gudula Schmidt,dagger Christian U. Lenzen, Heino Prinz, Dorothee Vogt, and Alfred Wittinghofer

Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany

Received 13 October 1998/Returned for modification 2 December 1998/Accepted 17 June 1999

The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras---a strong decrease in nucleotide affinity---enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.

* Corresponding author. Present address: Werkgroep Moleculaire Microbiologie, Rijksuniversiteit Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: 31 50 363 2158. Fax: 31 50 363 2154. E-mail: r.h.cool{at}biol.rug.nl.

dagger Present address: Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität, 79104 Freiburg, Germany.

Molecular and Cellular Biology, September 1999, p. 6297-6305, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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