Figure 2.

Rapamycin treatment alters the phosphorylation pattern of TIF-IA. (A) Two-dimensional tryptic phosphopeptide maps of TIF-IA labeled in vivo. HEK293T cells overexpressing Flag-tagged TIF-IA or TIF-IAS44A, respectively, were labeled for 2 h with [³²P]orthophosphate in the absence or presence of 20 nM rapamycin. TIF-IA was immunopurified and subjected to two-dimensional tryptic phosphopeptide mapping. (B) Nuclear extracts from rapamycin-treated cells fail to phosphorylate TIF-IA at S44. Immobilized TIF-IA was incubated with [γ-³²P]ATP and nuclear extract from untreated, rapamycin-treated, or amino acid-starved cells as indicated. The tryptic phosphopeptide map of in vitro phosphorylated TIF-IA was compared with the phosphorylation pattern of TIF-IA labeled in vivo.