Related Experiment Videos
Nitric oxide-stimulated guanine nucleotide exchange on p21ras
H M Lander1, J S Ogiste, S F Pearce
1Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA.
Abstract:
The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21ras is essential for NO-induced downstream signaling, such as NF-kappa B activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of the same cell. These studies identify p21ras as a target of NO in T cells and suggest that NO activates p21ras by an action which mimics that of guanine nucleotide exchange factors.
Insights
Nitric oxide (NO) directly activates the p21ras protein in human T cells by S-nitrosylation, mimicking guanine nucleotide exchange factors and enabling downstream signaling like NF-kappa B activation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Immunology
Background:
- The protooncogene p21ras is a key mediator of extracellular signal transduction.
- Understanding how p21ras is regulated is crucial for deciphering cellular responses.
Purpose of the Study:
- To investigate the effect of nitric oxide (NO) on p21ras activation in human T cells.
- To elucidate the mechanism by which NO activates p21ras.
- To determine the role of p21ras in NO-induced downstream signaling.
Main Methods:
- In vitro studies with purified recombinant p21ras.
- Circular dichroism analysis to assess conformational changes.
- Measurement of GTP-bound p21ras levels.
- Assessment of NF-kappa B activation.
Main Results:
- Nitric oxide (NO) directly and reversibly activates p21ras in human T cells, increasing GTP-bound p21ras.
- NO induces a significant conformational change in p21ras, facilitating GDP/GTP exchange.
- Activation occurs via S-nitrosylation of a critical cysteine residue on p21ras.
- p21ras is essential for NO-mediated activation of downstream signaling pathways, including NF-kappa B.
Conclusions:
- p21ras is a direct target of nitric oxide (NO) in T cells.
- NO activates p21ras through S-nitrosylation, a mechanism similar to guanine nucleotide exchange factors.
- This NO-induced p21ras activation is critical for downstream cellular responses in T cells.