Extended Data Fig. 7: CDK11 binds and phosphorylates the threonine-proline-rich N terminus of SF3B1.

a, Immunoblot analyses of immunoprecipitations of endogenous CDK11 in HEK293 cells. Detected proteins are indicated on the right. CK2a is known CDK11 interacting partner (ref. ²¹), IgG=antibody control. b, Immunoblot analyses of in vitro binding assays of GST-tagged SF3B1 (1-463) purified from E. coli and Flag-tagged CDK11 purified from HCT116 cells. Antibodies used are shown on the right. c, Schematic view of SF3B1 protein and its deletion mutants used in (d). d, Immunoblot analyses of IVKA using Flag-tagged AS CDK11 and Flag-tagged SF3B1, SF3B1 (1-463), and SF3B1 (464-1304) substrates. AS CDK11 remains catalytically active, but has mutated a gatekeeper methionine to glycine (M503G) in its catalytic site to allow the usage of thiophosphate ATP analogues (ref. ³⁸). This can be detected in substrates by western blotting using an anti-thiophosphate ester (TPE) antibody (ref. ⁵²). The blots were probed with Flag, CDK11, SF3B1 and TPE antibodies as indicated. Inputs represent aliquots of individual proteins added to the IVKA. e, Immunoblot of proteins after treatment of WT or SF3B1 R1074H HCT116 cells with 50 nM OTS964 for 3 min. f, Depiction of SF3B1 peptides found by IP-MS/MS in HCT116 cells with stably integrated Flag-tagged-SF3B1 treated with control DMSO or 50 nM OTS964 for 1 h; shown peptides were found in at least 3 replicates of control- but not OTS964-treated cells. Phosphorylated threonines and serines are shown in red; bold indicates the phosphorylated residues found in B^act complex (ref. ¹⁰). n = 4 replicates. g, Immunoblot of proteins after treatment of WT HCT116 cells with 20 nM dinaciclib for indicated times. h, Denaturing gel analyses of the radiolabelled AdML pre-mRNA and spliced products from in vitro splicing reactions in HeLa nuclear extracts treated with DMSO or 1 µM dinaciclib for indicated times (left panel). Native gel analyses of spliceosome assembly on radiolabelled AdML pre-mRNA in HeLa nuclear extracts treated with DMSO or indicated concentrations of dinaciclib for indicated times (right panel). See Fig. 3a, b for further legend.