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Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro
S Matsuoka1, G Rotman, A Ogawa
1Howard Hughes Medical Institute, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Abstract:
The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro, ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.
Insights
The protein kinase Chk2 is activated by DNA damage. Ataxia telangiectasia-mutated (ATM) phosphorylates Chk2 at Thr68 in response to ionizing radiation (IR), regulating its DNA damage response.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Chk2 is a key protein kinase in DNA damage response pathways.
- Its activation is crucial for cell cycle checkpoint control following DNA damage.
- The role of ATM in Chk2 activation varies depending on the type of DNA damage.
Purpose of the Study:
- To investigate the direct phosphorylation of Chk2 by ATM.
- To identify the specific phosphorylation sites on Chk2 involved in its activation.
- To elucidate the mechanism of Chk2 regulation by ATM in response to different DNA damaging agents.
Main Methods:
- In vitro kinase assays using purified ATM and Chk2.
- Site-directed mutagenesis of Chk2 phosphorylation sites.
- Analysis of Chk2 phosphorylation and activation in response to ionizing radiation (IR), UV irradiation, and hydroxyurea (HU).
Main Results:
- ATM directly phosphorylates Chk2 in vitro, with Thr68 being a major site within the SQ/TQ cluster domain (SCD).
- In vivo, Thr68 phosphorylation of Chk2 is ATM-dependent following IR but not UV or HU exposure.
- Mutation of Thr68 or all seven SQ/TQ motifs in Chk2 significantly impairs its phosphorylation and activation after IR.
Conclusions:
- Chk2 is directly phosphorylated by ATM in response to IR, highlighting a direct link in DNA damage signaling.
- Phosphorylation of the SCD, particularly at Thr68, is critical for Chk2 activation and function in the DNA damage response.
- Differential regulation of Chk2 activation by ATM underscores the complexity of cellular responses to various DNA damaging agents.