Figure 5.

PP2A counteracts S44 phosphorylation by Cdk2/cyclin E. (A) Dephosphorylation by PP2A inactivates TIF-IA. Immobilized Flag-tagged TIF-IA was incubated with 0.003 units of either PP2A (Upstate Biochemicals) or calf intestine alkaline phosphatase (CIAP, Roche Molecular Biochemicals) for 30 min at 30°C in the supplied phosphatase buffer. Thirty nanograms and 90 ng of untreated (lanes 2,3), PP2A-treated (lanes 4,5), and CIAP-treated (lanes 6,7) TIF-IA were assayed for their capability to activate transcription in nuclear extracts from rapamycin-treated cells. (B) Phosphatase inhibitors alleviate rapamycin-mediated inactivation of TIF-IA. TIF-IA was purified from cells that were treated for 1 h with either okadaic acid (1 μM), rapamycin (20 nM), or calyculin A (50 nM), in the absence or presence of rapamycin as indicated. Thirty nanograms (even numbers) and 90 ng (odd numbers except 1) of TIF-IA were assayed for their capability to activate transcription of nuclear extracts from rapamycin-treated cells. (C) Inhibition of PP2A/PP1 in vivo alleviates rapamycin-dependent hypophosphorylation of S44. Hela cells overexpressing Flag-tagged TIF-IA were labeled for 2 h with [³²P]orthophosphate in the absence (left) or presence (middle) of rapamycin. (Right) Metabolic labeling was performed in the presence of both rapamycin (20 nM) and calyculin (50 nM). Radiolabeled TIF-IA was immunopurified and subjected to two-dimensional tryptic phosphopeptide mapping.