Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro - PubMed (original) (raw)
Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro
S Matsuoka et al. Proc Natl Acad Sci U S A. 2000.
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.
Figures
Figure 1
Phosphorylation of Chk2 by ATM. (A) Chk2 phosphorylation by ATM from normal lymphoid cells. Immunoprecipitates with anti-ATM antibodies from normal lymphoblastoid cell lines (L40 and C3ABR) or AT lymphoblastoid cell lines (AT59 and L6) were incubated with [γ-32P]ATP and KD Chk2 fused to GST (GST-Chk2-KD) or p53-N47 (29). The control reaction was carried out without immunoprecipitate (No kinase). Proteins were resolved by SDS-PAGE and visualized by autoradiography. (B) Chk2 phosphorylation by transfected ATM. Immunoprecipitates with anti-FLAG from 293T cells transfected with the vector expressing FLAG-tagged wild-type ATM (ATM-WT) or kinase dead ATM (ATM-KD) were incubated with GST-Chk2-KD and [γ-32P]ATP. Proteins were resolved by SDS-PAGE and visualized by autoradiography or Western blotting with anti-FLAG antibodies.
Figure 2
Mapping ATM phosphorylation sites on Chk2. (A) Domain structure of human Chk2. SQ/TQ, Ser-Gln and Thr-Gln cluster domain; FHA, fork-head associated domain; Kinase, kinase domain; O, SQ and TQ motifs. (B) Phosphorylation of the SQ/TQ-cluster domain on Chk2 by ATM. Immunoprecipitates with anti-FLAG from 293T cells transfected with FLAG-ATM were incubated with [γ-32P]ATP and GST-SQ/TQ, GST-ΔSQ/TQ, or GST. Regions of SQ/TQ and ΔSQ/TQ fused to GST are shown in_A_. Proteins were resolved by SDS-PAGE and visualized by autoradiography.
Figure 3
Phosphorylation of Thr68 on Chk2 by ATM. (A) Immunoprecipitates with anti-FLAG from 293T cells transfected with the vector expressing FLAG-ATM were incubated with [γ-32P]ATP and wild-type GST-SQ/TQ or GST-SQ/TQ with Ala substitution at Ser19 (S19A), Thr26 (T26A), Ser28 (S28A), Ser33 (S33A), Ser35 (S35A), Ser50 (S50A), or Thr68 (T68A). Proteins were resolved by SDS-PAGE and visualized by autoradiography. (B) FLAG-ATM was incubated with [γ-32P]ATP and full-length “wild type” kinase dead Chk2 fused to GST (WT), GST-Chk2-KD with Ala substitution at Thr68 (T68A), or Ala substitution at all seven Thr and Ser in the N-terminal SQ/TQ motifs (7A). Proteins were resolved by SDS-PAGE and visualized by autoradiography. (C) FLAG-ATM was incubated with [γ-32P]ATP and GST-Chk2-KD2 (WT), a full-length “wild-type” kinase dead Chk2 fused to GST, or GST-Chk2-KD2 with Ala substitution at Ser357 (S357A), Thr432 (T432A), or at both Thr68 and Ser357 (T68A/S357A). Proteins were resolved by SDS-PAGE and visualized by autoradiography.
Figure 4
Phosphorylation of Thr68 in vivo. (A) Phosphorylation of Thr68 in response to DNA damage in vivo. 293T cells were untreated or treated with 20 Gy IR or 50 J/m2 UV radiation and harvested after 2 h, or grown in 1 mM HU for 24 h before harvesting. Chk2 protein immunoprecipitated with anti-Chk2 antibodies from these cells was immunoblotted with anti-phospho-Thr68 and anti-Chk2 antibodies. (B) 293T cells were transfected with plasmids expressing FLAG-tagged wild-type Chk2 (WT), or the T68A mutant Chk2 (T68A) and Chk2 proteins were immunoprecipitated with anti-FLAG antibody from untreated cells (−) or cells treated (+) with 20 Gy IR. Chk2 was immunoblotted with anti-phospho-T68 and anti-Chk2 antibodies. (C) ATM-dependent phosphorylation of Thr68 in response to IR. AT cells containing the vector alone (−ATM) and the same cells containing the vector expressing ATM (+ATM) were untreated or treated with 10 Gy IR or 50 J/m2 UV radiation and harvested after 1 h or were grown in 1 mM HU for 24 h before harvesting. Chk2 immunoprecipitated with anti-Chk2 antibodies from cells was immunoblotted with anti-phospho- and anti-Chk2 antibodies.
Figure 5
Phosphorylation of Chk2 by ATR. (A) FLAG-tagged wild-type ATR (ATR-WT) or kinase dead ATR (ATR-KD) was incubated with [γ-32P]ATP and wild-type GST-SQ/TQ (WT) or GST-SQ/TQ with Ala-substitution at Ser19 (S19A), Thr26 (T26A), Ser28 (S28A), Ser33 (S33A), Ser35 (S35A), Ser50 (S50A), or Thr68 (T68A). Proteins were resolved by SDS-PAGE and visualized by autoradiography. (B) FLAG-ATM or FLAG-ATR was incubated with [γ-32P]ATP and wild-type GST-SQ/TQ (WT), GST-SQ/TQ with Ala substitution at six Ser and Thr residues in SQ/TQ motifs except Ser19 (S19), Thr26 (T26), Ser28 (S28), Ser33 (S33), Ser50 (S50), or Thr68 (T68); or GST-SQ/TQ with Ala substitution at all seven Ser and Thr residues in SQ/TQ motifs (7A). Proteins were resolved by SDS-PAGE and visualized by autoradiography.
Figure 6
Regulation of Chk2 by phosphorylation of N-terminal SQ/TQ motifs. (A) Chk2−/− MEF cells infected with retroviruses encoding empty vector (Vector), wild-type human Chk2 (WT) or the Chk2 T68A mutant (T68A), or a mutant with Ala substitution at all seven N-terminal SQ/TQ motifs (7A) were not treated (−) or were treated (+) with 20 Gy ionizing radiation (IR) and harvested after 1 h. Protein from these cells was fractionated by SDS-PAGE and immunoblotted with anti-human Chk2 antibody. (B) Chk2 was immunoprecipitated with anti-Chk2 antibodies from cells in_A_. Immunoprecipitates were incubated with GST-Cdc25C () and [γ-32P]ATP. Proteins were resolved by SDS-PAGE and visualized by autoradiography or Western blotting with anti-human Chk2 antibodies.
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