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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
Molecular and Cellular Biology
|August 24, 1999
Summary
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.