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. 2022 Mar 16;12(3):210381. doi: 10.1098/rsob.210381

Figure 1.

Figure 1.

Cdk10 shares substrates with cell cycle and transcriptional CDKs. (a) Domain architectures of human Cdk10 (UniProt accession number Q15131) and human Cyclin Q (Q8N1B3). M117—gatekeeper methionine, T133—phosphorylation site, results in increased Cdk10 degradation, D163—catalytic centre, T196—conserved T-loop threonine phosphorylation is required for kinase activation. (b) Coomassie-stained SDS–PAGE analysis of 1 µg recombinant human Cdk10/CycQ. (c) Mass spectrometry-based detection of phosphorylation within recombinant human Cdk10 expressed in Sf9 insect cells. (d) Radiometric kinase assay; 0.2 µM Cdk10/CycQ, 25 µM GST-RB1 (761–928), 10 µM GST-CTD[52], 25 µM His-c-MYC (17–167) or 25 µM GST-SRSF7 (1–248) were incubated with 1 mM ATP containing 0.35 µCi γ[32P]-ATP for 30 min at 30°C. Data represent mean ± s.d. of duplicate measurements; cpm, counts per minute. (e) SDS–PAGE analysis (Coomassie) of 2 µg recombinant human MBP-Cdk10/His-CycQ, MBP-Cdk10T196A/CycQ and MBP-Cdk10T196E/CycQ. (f) Radiometric kinase assay of Cdk10 T-loop mutants. The experiments were performed similarly as in (d).