FIG. 4.

CAST/hPAF49 in Pol I is Tyr-phosphorylated in actively growing cells, whereas in serum-starved cells, in which promoter-specific Pol I transcription is downregulated, CAST/hPAF49 in Pol I is not detectably Tyr phosphorylated. (A) CAST/hPAF49 in Pol I is Tyr-phosphorylated in actively growing cells but not detectably in serum-starved cells. Nuclear extracts from actively growing (lanes a) or serum-starved (lane ss) HeLa cells (no serum for 24 h) transfected with an expression construct for Flag-tagged CAST/hPAF49 (WT or Y82F mutant) were immunoprecipitated with Flag-specific antibodies (Flag-IP), and immunocomplexes were extensively washed and eluted by Flag peptide. Proteins were immunoblotted and probed with antibodies specific for human A127, CAST/hPAF49, phosphotyrosine (P-Tyr), and PAF53. Lane 1 (In, input) is a marker for the Pol I subunits (DEAE 0.2 M fraction). (B) Downregulation of promoter-specific Pol I transcription in serum-starved cells. Immunoprecipitated and Flag peptide-eluted Pol I complexes from panel A (actively growing cells, WTa; serum-starved cells, WTss; and Y82F) were tested for nonspecific transcription activity (black) or for promoter-dependent transcription activity in a reconstituted transcription reaction with purified SL1 and recombinant UBF (gray). The data sets of panels A and B are of a representative experiment, which has been repeated twice. The activity associated with immunocomplexes was expressed as a percentage of that derived from actively growing cells (set at 100%), and experimental error bars have been included.