A kinase activity associated with simian virus 40 large T antigen phosphorylates upstream binding factor (UBF) and promotes formation of a stable initiation complex between UBF and SL1

Simian virus 40 large T antigen is a multifunctional protein which has been shown to modulate the expression of genes transcribed by RNA polymerase I (Pol I), II, and III. In all three transcription systems, a key step in the activation process is the recruitment of large T antigen to the promoter b y direct protein-protein interaction with the TATA binding protein (TBP)-TA F complexes, namely, SL1, TFIID, and TFIIIB, However, our previous studies on large T antigen stimulation of Pol I transcription also revealed that th e binding to the TBP-TAF(I) complex SL1 is not sufficient to activate trans cription. To further define the molecular mechanism involved in large T ant igen-mediated Pol I activation, we examined whether the high-mobility group box-containing upstream binding factor (UBF) plays any role in this proces s. Here, using cell labeling experiments, we showed that large T antigen ex pression induces an increase in UBF phosphorylation. Further biochemical an alysis demonstrated that UBF is phosphorylated by a kinase activity that is strongly associated with large T antigen, and that the carboxy-terminal ac tivation domain of UBF is required for the phosphorylation to occur, Using in vitro reconstituted transcription assays, we demonstrated that the inabi lity of alkaline phosphatase treated UBF to efficiently activate transcript ion can be rescued by large T antigen. Moreover, we showed that large T ant igen-induced UBF phosphorylation promotes the formation of a stable UBF-SL1 complex. Together, these results provide strong evidence for an important role for the large T antigen-associated kinase in mediating the stimulation of RNA Pol I transcription.