Figure 2. Dephosphorylation of in vivo labeled CDK9 by PP1.

(A) PP1 dephosphorylates CDK9 (³²P)-phosphorylated in cultured cells. 293T cells transfected with Flag-CDK9 vector were pulsed with (³²P) orthophosphate in the presence of 100 nM okadaic acid. Flag-CDK9 was immunoprecipitated with anti-Flag antibodies (lane 2) and subjected to dephosphorylation with PP1 (lanes 3 and 4), PP2A (lanes 5 and 6) or cdc25A (lanes 7 and 8). Lane 1, immunoprecipitation with non-specific mouse IgG. The reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager. (B) Comparison of phosphatase activities of PP1 and PP2A. Recombinant PP1 (0.1 U) and purified from human red blood cells PP2A (0.1 U) were assayed with the generic KR-pT-IRR substrate or phospho-Rb peptide and the reactions were stopped at indicated time points by the addition of malachite green solution. The amount of malachite green was quantified by the absorbance and recalculated into the phosphate concentration using phosphate standard curve. (C) Dephosphorylation of CDK9's Thr186 by PP1. 293T cells were transfected with vectors expressing Flag-CDK9 WT (lanes 1–4) or Flag-CDK9 T186A (lane 5) and treated at 48 hours posttransfection with 100 nM okadaic acid. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies, subjected to dephosphorylation with PP1 (lane 2) or PP2A (lane 4) and analyzed by immunoblotting with phospho-specific CDK9 Thr186 or anti-CDK9 antibodies. (D) Comparison of dephosphorylation of (³²P) CDK9 and Thr186-phosphorylated. Combined results from three independent experiments shown as percent of CDK9 dephosphorylation. *P<0.05. Results are from a typical experiment of 4 performed.