Fig. 2. ERK1/2 directly phosphorylate CDK12 on Thr548.

a HEK293 cells were transfected with Myc-tagged CDK12, serum-starved overnight, and stimulated for 10–30 min with PMA, 100 ng/ml; EGF, 25 ng/ml; Insulin, 100 nM or FBS,10%. Immunoprecipitated Myc-CDK12 was assayed for phosphorylation by immunoblotting with a phospho-motif antibody that recognizes the pS/T-P consensus motif. b, c HEK293 cells were transfected with Myc-tagged CDK12, serum-starved overnight, and treated with BVD-523 (2 µM) or PD184352 (10 µM), respectively, for 30 min before PMA stimulation for another 30 min. Immunoprecipitated Myc-CDK12 was assayed for phosphorylation by immunoblotting with a phospho-motif antibody that recognizes the pS/T-P consensus motif. d Schematic representation of human CDK12. RS arginine/serine-rich, PRM proline-rich motif, KD kinase domain. All potential ERK1/2 sites in CDK12 were predicted with high stringency using the ERK1 kinase motif of Scansite 4.0 (https://scansite4.mit.edu). Also shown is the representation of the four fragments of CDK12: F1 (aa 1–351), F2 (aa 352–711), F3 (aa 712–1101), and F4 (aa 1102–1484). e Recombinant active ERK1 was incubated with recombinant CDK12 (F1, F2, F3 or F4) in a kinase reaction with [γ-³²P]ATP. The resulting samples were subjected to SDS–PAGE, and the dried Coomassie-stained gel was autoradiographed. f Same as b, except that HEK293 cells were transfected with Myc-tagged CDK12 WT or unphosphorylable mutants (T525A, S534A, T548A and S681A) prior to overnight serum starvation and PMA stimulation (100 ng/ml) for 30 min. g Sequence alignment surrounding T548 in CDK12 from various species. h Same as f, except that cells were co-transfected with MEK-DD, an activated form of MEK1 (S218/222D), prior to being serum-starved overnight. i HEK293 cells were transfected with Myc-tagged CDK12 WT or an unphosphorylable mutant (T548A), serum-starved overnight, and stimulated with PMA (100 ng/ml) for 30 min. Immunoprecipitated Myc-CDK12 was then treated with 400 U λ-phosphatase for 30 min at 30 °C and CDK12 was assayed for phosphorylation by immunoblotting with a phosphospecific antibody targeted against Thr548. j Same as i, except that cells were treated with BVD-523 (2 µM) or PD184352 (10 µM) for 30 min prior to PMA stimulation (100 ng/ml) for 30 min. a–c, e, f, g–j Representative data of n = 3. Source data are provided as a Source Data file.