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Protein phosphatases regulate DNA-dependent protein kinase activity.
P Douglas1, G B Moorhead, R Ye
1Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
The Journal of Biological Chemistry
|May 29, 2001
Summary
Reversible protein phosphorylation regulates DNA-dependent protein kinase (DNA-PK) activity. Protein phosphatase 2A (PP2A) reactivates DNA-PK in cells, highlighting its role in DNA repair pathways.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- DNA-dependent protein kinase (DNA-PK) is crucial for DNA double-strand break repair via nonhomologous end joining.
- DNA-PK is essential for cellular responses to ionizing radiation and V(D)J recombination.
- Phosphorylation of DNA-PK subunits correlates with its kinase activity inactivation.
Purpose of the Study:
- To investigate the role of protein phosphatases in regulating DNA-PK kinase activity.
- To identify the specific protein phosphatase responsible for DNA-PK reactivation in vivo.
Main Methods:
- In vitro assays using purified DNA-PK, protein phosphatase 1, and protein phosphatase 2A (PP2A).
- Treatment of human lymphoblastoid cells with protein phosphatase inhibitors (okadaic acid, fostriecin).
- In vivo phosphorylation studies using [(32)P]inorganic phosphate labeling.
Main Results:
- Protein phosphatase 1 and PP2A catalytic subunits restored DNA-PK activity lost due to phosphorylation in vitro.
- PP2A-selective inhibitors (okadaic acid, fostriecin) significantly decreased DNA-PK activity in cells.
- In vivo, okadaic acid treatment led to increased phosphorylation of DNA-PK subunits.
Conclusions:
- Reversible protein phosphorylation is a key regulatory mechanism for DNA-PK kinase activity.
- A PP2A-like enzyme is likely responsible for reactivating DNA-PK in vivo.