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The Ras mutant D119N is both dominant negative and activated
R H Cool1, G Schmidt, C U Lenzen
1Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany. r.h.cool@biol.rug.nl
Abstract:
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.
Insights
Ras(D119N) mutation can inhibit cell function by sequestering guanine nucleotide exchange factors and binding GTP. Combining this with a second mutation creates a potent dominant-negative Ras mutant, crucial for understanding Ras signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Biochemistry
Background:
- Ras-like proteins are key regulators of cellular processes.
- Mutations in the NKxD nucleotide binding motif can alter Ras protein activity.
- Understanding Ras mutant function is critical for deciphering cell signaling pathways.
Purpose of the Study:
- To investigate the functional effects of the Ras D119N mutation in specific cell types.
- To elucidate the biochemical mechanisms underlying Ras D119N activity.
- To identify prerequisites for dominant-negative Ras mutant function.
Main Methods:
- Site-directed mutagenesis to introduce D119N mutation in Ras.
- Cell-based assays in PC12 and NIH 3T3 cells to assess Ras activity.
- Biochemical analyses to determine nucleotide binding affinity and protein-protein interactions.
Main Results:
- Ras(D119N) exhibits cell-specific inhibitory effects by reducing nucleotide affinity.
- The D119N mutation allows Ras to sequester guanine nucleotide exchange factors and bind GTP independently.
- Ras(D119N) can act as an activated Ras at high concentrations, but Ras(S17N) does not bind effectors.
- Introducing a second partial-loss-of-function mutation enhances Ras(D119N) into a strong dominant-negative mutant.
Conclusions:
- Dominant-negative Ras mutants require increased affinity for exchange factors over nucleotides and impaired effector interaction.
- Ras(D119N) function is concentration-dependent and influenced by its interaction with exchange factors.
- Dual mutations can convert Ras(D119N) into a potent dominant-negative inhibitor, impacting cellular processes like neurite outgrowth and DNA synthesis.