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Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain
Ashley Craig1, Mary Scott, Lindsay Burch
1Department of Molecular and Cellular Pathology, Cancer Research UK Laboratories, University of Dundee, UK.
Abstract:
The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21(WAF1) and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21(WAF1) at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.
Insights
Checkpoint kinase 2 (CHK2) recognizes tumor suppressor p53 through its DNA-binding domain. This interaction reveals novel docking sites and allosteric activation mechanisms for CHK2 signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Kinase Research
Background:
- The tumor suppressor p53 is a critical protein involved in DNA damage response.
- Checkpoint kinase 2 (CHK2) phosphorylates p53 at specific sites upon DNA damage.
- The precise mechanism of CHK2 recognition and activation by p53 has remained unclear.
Purpose of the Study:
- To elucidate the interaction between CHK2 and p53.
- To identify the specific domains of p53 involved in CHK2 recognition and activation.
- To investigate the allosteric regulation of CHK2 activity.
Main Methods:
- Peptide-based assays to map CHK2 binding sites on p53.
- Site-directed mutagenesis to analyze the role of specific amino acids in CHK2 docking.
- In vitro kinase assays to assess CHK2 activity on various substrates.
Main Results:
- Two peptides from the p53 DNA-binding domain were identified as CHK2 docking sites.
- These peptides stimulate CHK2 phosphorylation of full-length p53 and p53 fragments.
- CHK2 can be allosterically activated towards p21(WAF1) via a novel docking interaction.
Conclusions:
- The p53 DNA-binding domain contains critical sites for CHK2 recognition and activation.
- Novel allosteric mechanisms involving CHK2 docking sites regulate kinase activity.
- These findings reveal potential pathways for CHK2 signaling in vivo.