Identification of the human DEAD-box protein p68 as a substrate of Tlk1
Reinhard Kodym1, Christian Henöckl, Christoph Fürweger
1Department of Radiobiology, Clinic for Radiotherapy and Radiobiology, Medical University of Vienna, Vienna, Austria. reinhard.kodym@meduniwien.ac.at
Abstract:
The activity of the human protein kinase Tlk1 is down-regulated within minutes after exposure of cells to ionizing radiation. In order to identify signaling pathways which might be relevant in the radiation response of mammalian cells we screened nuclear proteins for substrates of Tlk1. Among several proteins one could be identified as p68 RNA helicase. Furthermore, it could be shown that Tlk1 phosphorylates immunoprecipitated p68. The phosphorylation of the C-terminal fragment of p68 by rTlk1 reduced its affinity to single stranded RNA in a gel shift assay. In addition, it could be demonstrated that increasing the Tlk1 activity in HT1080 cells by forced Tlk1 overexpression leads to an increased phosphorylation of endogenous p68, arguing that p68 might be a physiological substrate of Tlk1. These findings open the possibility that Tlk1 might participate in diverse biologic functions like cell growth and differentiation, pre-mRNA splicing, and transcriptional coactivation.
Insights
Human protein kinase Tlk1 activity decreases after radiation exposure. Researchers identified p68 RNA helicase as a Tlk1 substrate, suggesting Tlk1
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Human protein kinase Tlk1 (Tlk1) activity is rapidly reduced following ionizing radiation exposure in mammalian cells.
- Understanding the signaling pathways involved in the cellular radiation response is crucial.
Purpose of the Study:
- To identify nuclear protein substrates of Tlk1.
- To investigate the functional consequences of Tlk1-mediated phosphorylation of identified substrates.
Main Methods:
- Screening of nuclear proteins for Tlk1 substrates.
- In vitro phosphorylation assays using immunoprecipitated p68 RNA helicase.
- Gel shift assays to assess RNA binding affinity.
- Overexpression of Tlk1 in HT1080 cells to study endogenous p68 phosphorylation.
Main Results:
- p68 RNA helicase was identified as a substrate of Tlk1.
- Tlk1 phosphorylates p68, and this phosphorylation reduces its affinity for single-stranded RNA.
- Tlk1 overexpression increases endogenous p68 phosphorylation, supporting p68 as a physiological Tlk1 substrate.
Conclusions:
- Tlk1 phosphorylates p68 RNA helicase, potentially regulating its function.
- This interaction suggests Tlk1's involvement in radiation response pathways.
- Tlk1 may play roles in cell growth, differentiation, pre-mRNA splicing, and transcriptional coactivation.
