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.