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Activation of c-Ha-Ras by nitric oxide modulates survival responsiveness in neuronal PC12 cells
K K Teng1, D K Esposito, G D Schwartz
1Division of Hematology, Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021, USA.
Abstract:
p21(c-Ha-Ras) (Ras) can be activated by the guanine nucleotide exchange factor mSOS1 or by S-nitrosylation of cysteine 118 via nitric oxide (NO). To determine whether these two Ras-activating mechanisms modulate distinct biological effects, a NO-nonresponsive Ras mutant (Ras(C118S)) was stably expressed in the PC12 cells, a cell line that generates NO upon nerve growth factor treatment. We report here that Ras(C118S) functions indistinguishably from wild type Ras in activating and maintaining the mSOS1- and Raf-1-dependent mitogen-activated protein kinase cascade necessary for neuronal differentiation. However, continuous (>5 days) exposure to nerve growth factor reveals that, in contrast to parental or wild-type Ras-overexpressing PC12 cells, Ras(C118S)-expressing PC12 cells cannot sustain the basal interaction between Ras and phosphatidylinositol 3-kinase. This results in spontaneous apoptosis of these cells despite the presence of nerve growth factor and serum. Thus unique downstream effector interactions and biological outcomes can be differentially modulated by distinct modes of Ras activation.
Insights
Distinct Ras activation pathways lead to different biological outcomes. While both pathways support neuronal differentiation, nitric oxide-independent Ras fails to prevent apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Neuroscience
Background:
- Ras proteins are key regulators of cellular signaling pathways.
- Ras activation can occur through various mechanisms, including guanine nucleotide exchange factors and post-translational modifications like S-nitrosylation.
- Nitric oxide (NO) mediates S-nitrosylation of Ras at cysteine 118.
Purpose of the Study:
- To investigate whether distinct Ras activation mechanisms (mSOS1-mediated vs. NO-mediated S-nitrosylation) lead to different biological effects.
- To determine the role of NO-mediated Ras activation in neuronal differentiation and survival.
Main Methods:
- Stable expression of a NO-nonresponsive Ras mutant (Ras(C118S)) in PC12 cells.
- Treatment of PC12 cells with nerve growth factor (NGF).
- Analysis of the mitogen-activated protein kinase (MAPK) cascade activation and Ras-effector interactions (Raf-1, phosphatidylinositol 3-kinase [PI3K]).
- Assessment of cell survival and apoptosis.
Main Results:
- Ras(C118S) mutant protein functions similarly to wild-type Ras in activating the mSOS1- and Raf-1-dependent MAPK cascade, supporting neuronal differentiation.
- Continuous NGF exposure revealed that Ras(C118S)-expressing cells failed to maintain Ras interaction with PI3K.
- This impaired PI3K interaction led to spontaneous apoptosis in Ras(C118S) cells, despite NGF and serum presence.
Conclusions:
- Distinct modes of Ras activation differentially modulate downstream effector interactions and biological outcomes.
- NO-mediated Ras activation is crucial for maintaining Ras-PI3K interaction, which is essential for long-term cell survival.
- This highlights a unique role for NO signaling in Ras-mediated cell survival beyond its role in differentiation.