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Published on: June 9, 2020
RNF144A, an E3 ubiquitin ligase for DNA-PKcs, promotes apoptosis during DNA damage
Shiuh-Rong Ho1, Christina S Mahanic2, Yu-Ju Lee3
1Section of Hematology/Oncology, Department of Medicine.
Abstract:
Several ring between ring fingers (RBR) -domain proteins, such as Parkin and Parc, have been shown to be E3 ligases involved in important biological processes. Here, we identify a poorly characterized RBR protein, Ring Finger protein 144A (RNF144A), as the first, to our knowledge, mammalian E3 ubiquitin ligase for DNA-PKcs. We show that DNA damage induces RNF144A expression in a p53-dependent manner. RNF144A is mainly localized in the cytoplasmic vesicles and plasma membrane and interacts with cytoplasmic DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). DNA-PKcs plays a critical role in the nonhomologous end-joining DNA repair pathway and provides prosurvival signaling during DNA damage. We show that RNF144A induces ubiquitination of DNA-PKcs in vitro and in vivo and promotes its degradation. Depletion of RNF144A leads to an increased level of DNA-PKcs and resistance to DNA damaging agents, which is reversed by a DNA-PK inhibitor. Taken together, our data suggest that RNF144A may be involved in p53-mediated apoptosis through down-regulation of DNA-PKcs when cells suffer from persistent or severe DNA damage insults.
Insights
Ring Finger protein 144A (RNF144A) is identified as a novel E3 ubiquitin ligase targeting DNA-PKcs. This finding suggests RNF144A
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- Ring Between Ring (RBR) domain proteins function as E3 ligases in cellular processes.
- DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is crucial for DNA repair and cell survival.
- The role of Ring Finger protein 144A (RNF144A) in DNA damage response is largely unknown.
Purpose of the Study:
- To identify novel E3 ubiquitin ligases involved in DNA damage response.
- To characterize the function of RNF144A in the context of DNA damage.
- To elucidate the regulatory mechanism of DNA-PKcs stability.
Main Methods:
- Protein expression analysis and western blotting.
- Immunoprecipitation and co-localization studies.
- In vitro and in vivo ubiquitination assays.
- RNA interference and pharmacological inhibition.
Main Results:
- RNF144A expression is induced by DNA damage in a p53-dependent manner.
- RNF144A interacts with cytoplasmic DNA-PKcs and promotes its ubiquitination and degradation.
- RNF144A depletion increases DNA-PKcs levels and confers resistance to DNA damaging agents.
- RNF144A-mediated DNA-PKcs down-regulation is reversed by DNA-PK inhibitors.
Conclusions:
- RNF144A acts as a mammalian E3 ubiquitin ligase for DNA-PKcs.
- RNF144A may mediate p53-dependent apoptosis by down-regulating DNA-PKcs under severe DNA damage.
- RNF144A represents a potential therapeutic target for modulating DNA damage response.
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