Figure 4

CDK2 phosphorylates Ser90 residue of CDK9. (A) CDK2 phosphorylates Ser90-containing peptide. CDK9-derived chemically synthesized peptides containing Thr29; Ser175 and Thr186; or Ser90 residues were phosphorylated by recombinant CDK2/cyclin E and analyzed by Phosphor Imaging Device (upper panel) or stained with Coomassie (lower panel). (B) CDK9 S90A mutant is less phosphorylated by CDK2/cyclin E in vitro. WT CDK9 and CDK9 S90A were expressed in 293T cells, precipitated with anti-FLAG antibodies and incubated with recombinant CDK2/cyclin E in the presence of (³²P) ATP. The reactions were resolved on a 10% SDS Tris-glycine gel and analyzed by immunoblotting (upper panel) or on Phosphor Imaging Device (lower panel). Quantification from the Phosphor Imager is shown. (C) Substitution of CDK9 Ser 90 prevents its phosphorylation in cultured cells. 293T cells transfected with FLAG-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1, pulse-labeled with (³²P) and CDK9 was immunoprecipitated from cellular lysates with anti-FLAG antibodies, and analyzed by immunoblotting (upper panel) or by Phosphor Imaging Device (lower panel). Quantification of the bands on Phosphor Imager is shown for three independent experiments. (D and E) CDK9 Ser90 phosphorylation is decreased in CDK2 KD cells. 293T cells were transfected with vectors expressing Flag-CDK9 WT or Flag-CDK9 S175A (panel D); or 293T and 293T-59 cells were transfected with a vector expressing Flag-CDK9 WT (panel E) and after 48 hrs in culture treated with 0.1 μM okadaic acid. CDK9 was immunoprecipitated with anti-Flag antibodies and analyzed by immunoblotting with anti-CDK9 or Ser90 phospho-specific antibodies.