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Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a
Myoung-Ae Kim1, Hyun-Ju Kim, Alexandra L Brown
1Medical Research Center for Cancer Molecular Therapy, College of Medicine, Dong-A University, Busan 602-714, Korea.
Abstract:
Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1a were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1a were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.
Insights
Researchers identified new proteins targeted by Checkpoint kinase 1 (Chk1) and Chk2, crucial enzymes in DNA damage response. This discovery advances understanding of their tumor suppressor roles.
Area of Science:
- Molecular Biology
- Cellular Biology
- Oncology
Background:
- Checkpoint kinase 1 (Chk1) and Chk2 are key regulators of the DNA damage response.
- Dysfunctional Chk1/Chk2 activity is linked to cancer development.
- Identifying novel substrates is crucial for understanding their tumor suppressor mechanisms.
Purpose of the Study:
- To identify novel in vitro substrates of Chk1 and Chk2.
- To deepen the understanding of Chk1 and Chk2 functions in DNA damage response and cancer.
Main Methods:
- Utilized previously determined substrate target motifs for screening.
- Performed genome-wide peptide database searches for candidate identification.
- Validated potential substrates using in vitro kinase assays and immunoprecipitation kinase assays.
Main Results:
- Identified ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D, and MAD1a as novel in vitro substrates for Chk1 and/or Chk2.
- Further analysis confirmed HDAC5 and PGC-1alpha as specific targets of Chk1 and/or Chk2 in vitro.
- Site-directed mutagenesis validated the identified target motifs.
Conclusions:
- Successfully identified multiple novel substrates for Chk1 and Chk2.
- The findings expand the known interactome of Chk1 and Chk2.
- This research contributes to understanding the tumor suppressor functions of Chk1 and Chk2.
