Fig. 5: HIPKs phosphorylate general transcription factors and RNA pol II CTD.
From: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation
a For radioactive kinase activity assays 10 μM human SPT5, His-c-Myc, GST-CTD[52], and GST as a control were incubated with 0.2 mM [32P]-γ-ATP either with or without 0.2 μM kinase for 30 min. b For in vitro kinase assays 10 μM GST-CTD[52], 0.2 mM ATP, and 0.2 μM kinase were incubated for 120 min, and visualized by immunoblotting using RNA pol II CTD specific phosphoantibodies pTyr1 (3D12), pSer2 (3E10), pThr4 (6D7), pSer5 (3E8), and pSer7 (4E12). c Gel shift assays were performed as in (b) but in time-course series and visualized by Coomassie staining in SDS gels. Upon phosphorylation the GST-CTD[52] band shifts from the IIa to the IIo form. d In vitro kinase assays were performed as in (b) but as time-course series, visualized by immunoblotting. e Radioactive kinase assays were performed as in (a) using 150 μM pre-phosphorylated CTD Peptides as a substrate. Each CTD peptide contained three consensus hepta-repeats with either no modification (cons. CTD) or with phosphorylation marks continuously set at one residue of the heptad sequence as depicted in the cartoon. The principle was used for Tyr1, Ser2, Thr4, Ser5, Ser7 or a peptide that contained a lysine at position 7. Measurements were performed as duplicates (n = 2 biologically independent samples) and are depicted as mean. Source data are provided as a Source Data file.
