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. 2009 Jun 18;28(24):2314-23.
doi: 10.1038/onc.2009.102. Epub 2009 May 4.

Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity

M Walker  1 , E J Black, V Oehler, D A Gillespie, M T Scott
Affiliations

Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity

M Walker et al. Oncogene. .

Abstract

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ATR kinases during checkpoint activation; however, how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell-cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345 and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/M and S/M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, as the mutants are either partially (317A and 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated by a de-repression mechanism.

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Figures

Figure 1
Figure 1. Activation and phosphorylation of Chk1 in response to irradiation and DNA synthesis inhibition
(A) Kinase activity of Chk1 immunoprecipitated from wild type DT40 (WT) cells treated with 60 μM aphidicolin or 10Gy IR. Shown is the mean +/− S.E.M of ≥ 4 independent experiments. Immunoprecipitation and lysis conditions are described in detail in Supplementary methods. (B, C) Western blot analysis of cell lysates from the aphidicolin or IR treated cultures using anti-S317, anti-S345 and anti-S366 phospho-specific Chk1 antibodies plus anti Chk1 MAb (G-4). (D) Western blot and autoradiograph of Chk1 immunoprecipitates shown in (A) analysed post kinase assay.
Figure 2
Figure 2. Cell cycle phase specificity of Chk1 activation and phosphorylation
(A) Percentage of DT40 cells in each phase of cell cycle before (L) and after fractionation of an asynchronous culture by centrifugal elutriation, as determined by flow cytometry analysis of DNA content, BrdU incorporation and pS10 histone H3 positive mitotic cells. Centrifugal elutriation was performed as described previously (Zachos et al., 2005) and in Supplementary methods. (B, C, and D) Kinase activity of Chk1 immunoprecipitated from untreated (B), aphidicolin treated (C), or irradiated (D) cells before and after fractionation. Values represent mean +/− S.E.M of four independent experiments Extracts assayed for Chk1 kinase activity (left panels) were also analysed by western blotting (right panels) using anti-S317, anti-S345 and anti-S366 phospho-specific antibodies and anti Chk1 MAb (G-4).
Figure 3
Figure 3. Determination of G2/M and S-M checkpoint proficiency of Chk1 SQ site mutants
(A) Chk1 null cells were stably reconstituted with either WT Chk1 or mutants lacking individual SQ phosphorylation sites as shown in the schematic. (B) Lysates from each cell line were analysed by western blotting using antibodies specific for Chk1 and actin as a loading control. (C) G2/M checkpoint proficiency was determined by quantifying the accumulation of pS10 histone H3 positive mitotic cells in cultures treated with either nocodozole alone or IR (10 Gy) plus nocodozole for 10 hours. Black bars represent the number of mitotic cells accumulated in the irradiated culture expressed as a percentage of the nocodazole control. S-M checkpoint proficiency was assessed by treating each cell line with aphidicolin plus nocodozole for 10 hours and quantifying both the total percentage of mitotic cells (grey bars) and the percentage of pS10 histone H3 positive cells with 2N DNA content (premature mitosis: red bars). Flow cytometry was performed as described previously (Zachos et al., 2005) and in Supplementary methods.
Figure 4
Figure 4. Phosphorylation of S345 is essential for activation of Chk1 and dependent on modification of additional adjacent SQ sites
(A, B) Kinase activity of Chk1 immunoprecipitated from DT40 and SQ site mutant-expressing DT40 cells over a three hour time course following treatment with either 60 μM aphidicolin (A) or 10Gy IR (B). Shown is the mean +/− S.E.M of four independent experiments. (C, D) Western blot analysis of lysates from selected cell lines shown in (A) treated for 15 mins with aphidicolin or 10Gy IR using either anti-S317, anti-S345, anti-S366 (panel C only) phospho-specific Chk1 antibodies, or Chk1 MAb (G4).
Figure 5
Figure 5. Chk1 SQ site phosphorylation relieves repression of latent kinase activity
(A) Chk1 was immunoprecipitated from DT40 cells treated with and without aphidicolin for 30 mins and the precipitates washed with either lysis buffer (LB) or RIPA buffer before being analysed by western blotting (upper panel) or subjected to in vitro kinase assay followed by western blotting (middle panel) and autoradiography (lower panel). (B) Kinase activity of replicate immunoprecipitates as shown in (A) determined using Chktide substrate, results are mean +/− S.E.M of three independent experiments. (C) Kinase activity against Chktide substrate of Chk1 immunoprecipitated from DT40, KD- and S345A-expressing cell lines, where precipitates had been washed with either LB or RIPA as in (A). Results are mean +/− S.E.M of three independent experiments. (D) Cos1 cells were co-transfected with Chk1 Δ400 mutant minus and plus increasing amounts of a plasmid encoding the Chk1 C-terminal regulatory domain (amino acids 260-476) for 24 hours. Extracts were analysed by western blotting using anti-Chk1 MAb (upper panel) or anti C-terminal Chk1 (middle panel). Chk1 260-476 was immunoprecipitated from the lysates using C-terminal Chk1 Ab and co-precipitation of Chk1 Δ400 verified by western blotting with Chk1 MAb (lower panel). (E) Cos1 cells were transfected with plasmids encoding Chk1 Δ400 minus and plus Chk1 260-476. Chk1 260-476 was immunoprecipitated using C-terminal antiserum and the precipitates washed with either LB or RIPA as in (A) before being analysed for Chk1 Δ400 by western blotting using Chk1 MAb.
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