Figure 4

In vitro validation of selective candidate CDK2 substrates. (a) HEK293 cells were transiently transfected with vectors expressing FLAG-tagged EF2, TRF2, and RAP1. Anti-FLAG antibody immunoprecipitates were in vitro phosphorylated with cyclin A-CDK2 or cyclin B-CDK1 in the presence of γ-³²P-ATP (upper left panels). In parallel reactions, histone H1 was phosphorylated as a control to normalize the activities of cyclin A-CDK2 and cyclin B-CDK1 (right panel). 'C' denotes 'kinase only' reactions without transfected substrates (left panel) and 'no kinase' reaction (right panel). Protein samples were separated by SDS PAGE and the gels were transferred onto PVDF membranes. Phospho-signals were visualized by autoradiography. The membrane was subsequently probed with anti-FLAG antibody (Sigma-Aldrich) to confirm the identity of the phospho-signal bearing band (lower left panels). The asterisk represents a non-specific band from the commercial cyclin B-CDK2 preparation. (b) Kinase reaction was carried out using γ-³²P-ATP, and GST-TRF2, GST-RAP1, GST-Rb (positive control) and GST (negative control) as substrates in the presence or absence of wild-type cyclin A-CDK2 kinase. Reactions were visualized by SDS PAGE followed by Coomassie staining and autoradiography (left panel). A similar kinase assay was carried out using wild-type cyclin A/CDK2 and ATP-γ-S, and subsequently subjected to a phosphopeptide isolation scheme. MS analysis confirmed that TRF2 and RAP1 were each phosphorylated on the exact sites we identified from the screen with one additional site for RAP1 (right panel). (c) Kinase assay was carried out using γ-³²P-ATP, cyclin A-CDK2 or cyclin B-CDK1 with increasing amounts of purified GST-RL12. Samples were separated by SDS PAGE and the gel was stained with Coomassie (lower panel) followed by autoradiography (upper panel). 'C' denotes 'kinase only' reactions without transfected substrates.