Related Experiment Videos
jun-NH2-terminal kinase activation mediated by UV-induced DNA lesions in melanoma and fibroblast cells
1Molecular Carcinogenesis Program, American Health Foundation, Valhalla, New York 10595, USA.
Abstract:
jun-NH2-terminal kinase (JNK) belongs to a family of protein kinases that phosphorylates c-Jun, ATF2, and Elk1 in response to various forms of stress including UV irradiation and heat shock. Although in previous studies we have demonstrated the importance of membrane components for JNK activation by UV irradiation, here we have elucidated the role of DNA damage in this response. We show that in vitro-irradiated or sonicated DNA that is added to proteins prepared from UV-treated cells can further induce JNK activation in a dose-dependent manner. When compared with UV-B (300 nm), UV-C (254 nm), which is better absorbed by the DNA, is significantly more potent in activating JNK. Furthermore, when wavelengths lower than 300 nm were filtered out, UV-B was no longer able to activate JNK. With the aid of melanoma and fibroblast cells, which exhibit different resistances to irradiation and require different UV doses to generate the same number of DNA lesions, we demonstrate that above a threshold level of 0.45 lesions and up to 0.75 lesions per 1875 bp, the degree of JNK activation correlates with the amount of lesions induced by UV-C irradiation. Finally, to explore the role of nuclear and mitochondrial DNA (mtDNA) in mediating JNK activation after UV irradiation, we have used cells that lacks mtDNA. Although the lack of mtDNA did not impair the ability of UV to activate JNK, when enucleated, these cells had lost the ability to activate JNK in response to UV irradiation. Overall, our results suggest that DNA damage in the nuclear compartment is an essential component that acts in concert with membrane-anchored proteins to mediate c-Jun phosphorylation by JNK.
Insights
DNA damage, particularly in the nucleus, is crucial for activating jun-NH2-terminal kinase (JNK) in response to UV radiation. This process works alongside membrane proteins to phosphorylate c-Jun.
Area of Science:
- Molecular Biology
- Cell Signaling
- DNA Damage Response
Background:
- Jun-NH2-terminal kinase (JNK) is a protein kinase activated by cellular stress, including UV irradiation.
- Previous research highlighted the role of membrane components in UV-induced JNK activation.
- The specific contribution of DNA damage to this signaling pathway remained to be fully elucidated.
Purpose of the Study:
- To investigate the role of DNA damage in mediating jun-NH2-terminal kinase (JNK) activation following UV irradiation.
- To determine whether nuclear or mitochondrial DNA is primarily involved in UV-induced JNK signaling.
- To establish the relationship between the extent of DNA lesions and the degree of JNK activation.
Main Methods:
- In vitro experiments using irradiated/sonicated DNA and cell extracts to assess JNK activation.
- Comparative analysis of JNK activation using different UV wavelengths (UV-B vs. UV-C) and filtered light.
- Utilizing melanoma and fibroblast cells with varying UV sensitivities to correlate DNA lesions with JNK activation.
- Employing mtDNA-lacking cells and enucleated cells to differentiate the roles of nuclear and mitochondrial DNA.
Main Results:
- Irradiated or sonicated DNA directly induced JNK activation in a dose-dependent manner.
- UV-C (254 nm), which is better absorbed by DNA, was more potent in JNK activation than UV-B (300 nm).
- JNK activation correlated with the number of DNA lesions induced by UV-C irradiation within a specific range.
- UV-induced JNK activation was abolished in enucleated cells but unaffected by the absence of mtDNA.
Conclusions:
- Nuclear DNA damage is a critical factor in mediating UV-induced JNK activation.
- This DNA damage acts in concert with membrane-associated proteins to regulate JNK signaling.
- Mitochondrial DNA does not appear to play a significant role in this specific UV-response pathway.