Product Pathways - DNA Damage
Chk2 Kinase #7434
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Description
Purified recombinant full length human Chk2 (Met1-Leu543) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human Chk2 (Met1-Leu543) (GenBank Accession No. NM_007194) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-Chk2 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. Chk2 kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Chktide 400 ng/μL and recombinant Chk2: variable.
Quality Control
The theoretical molecular weight of the GST-Chk2 fusion protein is 88 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. Chk2 kinase activity was determined using a radiometric assay [Fig.2].
Background
Chk2 is the mammalian orthologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (1-3). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50 and Thr68) each followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (4,5). After DNA damage by ionizing radiation (IR), UV irradiation or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (5-7). The SQ/TQ cluster domain, therefore, seems to have a regulatory function. Phosphorylation at Thr68 is a prerequisite for the subsequent activation step, which is attributable to autophosphorylation of Chk2 on residues Thr383 and Thr387 in the activation loop of the kinase domain (8).
- Allen, J.B. et al. (1994) Genes Dev. 8, 2401-2415.
- Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665.
- Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
- Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
- Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
- Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
- Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.
- Lee, C.H. and Chung, J.H. (2001) J. Biol. Chem. 276, 30537-30541.
Application References
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