Samenvatting
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 effecters. 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.
| Originele taal-2 | English |
|---|---|
| Pagina's (van-tot) | 6297-6305 |
| Aantal pagina's | 9 |
| Tijdschrift | Molecular and Cellular Biology |
| Volume | 19 |
| Nummer van het tijdschrift | 9 |
| DOI's | |
| Status | Published - sep.-1999 |
| Extern gepubliceerd | Ja |
Toegang tot document
Handle.net
The Ras Mutant D119N Is Both Dominant Negative and Activated
Final publisher's version, 153 KB
Kopie aanvragen
Document onder embargo
Citeer dit
- APA
- Author
- BIBTEX
- Harvard
- Standard
- RIS
- Vancouver
Cool, RH., Schmidt, G., Lenzen, CU., Prinz, H., Vogt, D., & Wittinghofer, A. (1999). The Ras mutant D119N is both dominant negative and activated. Molecular and Cellular Biology, 19(9), 6297-6305. https://doi.org/10.1128/MCB.19.9.6297
Cool, RH ; Schmidt, G ; Lenzen, CU et al. / The Ras mutant D119N is both dominant negative and activated. In: Molecular and Cellular Biology. 1999 ; Vol. 19, Nr. 9. blz. 6297-6305.
@article{51f979d946ba4a189ef8fe519839e9c3,
title = "The Ras mutant D119N is both dominant negative and activated",
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 effecters. 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.",
keywords = "NUCLEOTIDE-EXCHANGE FACTOR, GDP DISSOCIATION STIMULATOR, C-FOS PROMOTER, NIH 3T3 CELLS, HA-RAS, SACCHAROMYCES-CEREVISIAE, MAP KINASE, SIGNAL-TRANSDUCTION, ONCOGENIC RAS, GROWTH-FACTOR",
author = "RH Cool and G Schmidt and CU Lenzen and H Prinz and D Vogt and A Wittinghofer",
year = "1999",
month = sep,
doi = "10.1128/MCB.19.9.6297",
language = "English",
volume = "19",
pages = "6297--6305",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "AMER SOC MICROBIOLOGY",
number = "9",
}
Cool, RH, Schmidt, G, Lenzen, CU, Prinz, H, Vogt, D & Wittinghofer, A 1999, 'The Ras mutant D119N is both dominant negative and activated', Molecular and Cellular Biology, vol. 19, nr. 9, blz. 6297-6305. https://doi.org/10.1128/MCB.19.9.6297
The Ras mutant D119N is both dominant negative and activated. / Cool, RH; Schmidt, G; Lenzen, CU et al.
In: Molecular and Cellular Biology, Vol. 19, Nr. 9, 09.1999, blz. 6297-6305.
Onderzoeksoutput › Academic › peer review
TY - JOUR
T1 - The Ras mutant D119N is both dominant negative and activated
AU - Cool, RH
AU - Schmidt, G
AU - Lenzen, CU
AU - Prinz, H
AU - Vogt, D
AU - Wittinghofer, A
PY - 1999/9
Y1 - 1999/9
N2 - 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 effecters. 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.
AB - 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 effecters. 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.
KW - NUCLEOTIDE-EXCHANGE FACTOR
KW - GDP DISSOCIATION STIMULATOR
KW - C-FOS PROMOTER
KW - NIH 3T3 CELLS
KW - HA-RAS
KW - SACCHAROMYCES-CEREVISIAE
KW - MAP KINASE
KW - SIGNAL-TRANSDUCTION
KW - ONCOGENIC RAS
KW - GROWTH-FACTOR
U2 - 10.1128/MCB.19.9.6297
DO - 10.1128/MCB.19.9.6297
M3 - Article
C2 - 10454576
SN - 0270-7306
VL - 19
SP - 6297
EP - 6305
JO - Molecular and Cellular Biology
JF - Molecular and Cellular Biology
IS - 9
ER -
Cool RH, Schmidt G, Lenzen CU, Prinz H, Vogt D, Wittinghofer A. The Ras mutant D119N is both dominant negative and activated. Molecular and Cellular Biology. 1999 sep.;19(9):6297-6305. doi: 10.1128/MCB.19.9.6297
FAQs
What is the dominant negative form of Ras? ›
Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling.
Is Ras mutation dominant? ›The use of the dominant negative mutant of Ras has been crucial in elucidating the cellular signaling of Ras in response to the activation of various membrane-bound receptors.
What does a dominant negative mutation mean? ›Dominant-negative is a term used to describe a type of genetic mutation that results in a protein that interferes with the normal function of the wild-type protein. This occurs when the mutant protein product, often non-functional, competes with the standard protein product for binding to other cellular components.
What is an example of a dominant negative mutation? ›A well-known example of a gene that can incur dominant-negative mutations is the small GTPase Ras. These dominant-negative alleles of Ras are not functional themselves because they preferentially bind GDP and stay locked in the inactive state.
Is RAS dominant or recessive? ›Because this type of mutation makes a gene product hyperactive, the effect is dominant—only one of the cell's two gene copies needs to undergo the change. The Ras genes are mutated in a wide range of human cancers, and they remain one of the most important examples of cancer-critical genes.
Which is the active form of the RAS protein quizlet? ›The Ras protein is a GTPase that functions in many growth factor signaling pathways. In its active form, with GTP bound, it transmits a downstream signal that leads to cell proliferation; in its inactive form, with GDP bound, the signal is not transmitted.
What does RAS mutation mean? ›When RAS genes are mutated, cells grow uncontrollably and evade death signals. RAS mutations also make cells resistant to some available cancer therapies.
How is Ras gene activated? ›RAS is activated by GDP/GTP exchange stimulated by GEFs and inactivated by GTP hydrolysis stimulated by GAPs. The very slow off-rate for GDP (t1/2 = 6 min, koff = 2 × 10−3 s−1 at 20°) (Hunter et al., 2015) allows RAS proteins to remain in their inactive states until signals provoke GDP/GTP exchange.
What happens when RAS is activated? ›The activated ras protein acts as a molecular switch that turns on various target proteins necessary for important cellular processes such as division and proliferation. In normal cells, a balanced cycling of the GTP to GDP through the inherent GTPase activity of ras keeps ras-mediated signaling in check.
Is dominant positive or negative? ›While a dominant personality is often associated with negative traits of aggression and manipulation, there are many positive traits to be recognized, as well. Dominant people can be strong leaders, dedicated, and goal-oriented.
What does dominant mutation mean? ›
Dominant gene variants are misspellings (mutations) that occur in one copy of the gene pair while the other copy of the gene is spelled correctly. Dominant variants can cause HI in a person even if the other copy of the gene is normal.
What is the difference between a positive and negative mutation? ›Based on their effects on fitness, mutations can be divided into three broad categories: the 'good' or advantageous that increase fitness, the 'bad' or deleterious that decrease it and the 'indifferent' or neutral that are not affected by selection because their effects are too small.
What causes dominant mutation? ›Individuals receive two versions of each gene, known as alleles, from each parent. If the alleles of a gene are different, one allele will be expressed; it is the dominant gene. The effect of the other allele, called recessive, is masked.
Which mutations are most damaging? ›Mutations caused by insertion or deletion of nitrogenous bases in the DNA sequence leads to the most damage to DNA since it completely changes the reading frame for the process of protein synthesis. Insertion or deletion that results in frameshift mutation are considered as the most damaging mutation.
Are most mutations positive or negative? ›Most mutations are not harmful, but some can be. A harmful mutation can result in a genetic disorder or even cancer. Another kind of mutation is a chromosomal mutation.
What percentage of cancers have RAS mutations? ›We estimate that approximately 19% of patients with cancer will harbor a Ras mutation; this is equivalent to approximately 260,000 new cases per year in the United States. Globally, there are currently approximately 18 million new cancer diagnoses per year (48).
What percent of cancers show mutations to RAS? ›RAS mutations (HRAS, NRAS, and KRAS) are among the most common oncogenes, and around 19% of patients with cancer harbor RAS mutations.
Which mutant form of RAS is likely to cause malignancy? ›Most cancer types favour mutation of a single isoform; this is typically K-Ras.
What activates Ras signaling? ›RAS signaling can be activated by a number of cellular receptors including receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and integrin family members.
What is the ras gene quizlet? ›define ras gene. a gene that codes for ras protein a g protein that relays a growth signal from a growth factor or receptor on the plasma membrane to a cascade of protein kinases that ultimately results in the stimulation of the cell cycle.
What is Ras activation followed by quizlet? ›
Ras activation is followed by: hormone binding to the receptor.
What is a dominant negative inhibitor? ›Dominant negative inhibition is a phenomenon in which the function of a wild-type gene product is impaired by a coexpressed mutant variant of the same gene product (1).
What are the forms of RAS proteins? ›All mammalian cells express three closely related Ras proteins: H-Ras, K-Ras and N-Ras that promote oncogenesis when mutationally activated at codons 12, 13 or 61.
What are the different types of RAS mutations? ›Five mutations (G12D, G12V, G12C, G13D, and Q61R) account for 70% of all Ras-mutant patients. G12C mutations are frequently found in lung cancer due to G:C>T:A transversions associated with bulky adducts generated by the mutagens in tobacco smoke (51).
What are the different types of Ras gene? ›The main members of the RAS gene family— KRAS, HRAS, and NRAS—encode proteins that have a pivotal cytoplasmic role in cell signaling. When RAS genes are mutated, cells grow uncontrollably and evade death signals. RAS mutations also make cells resistant to some available cancer therapies.