The putative cofactor TIF1α is a protein kinase that is hyperphosphorylated upon interaction with liganded nuclear receptors

Ligand-induced gene activation by nuclear receptors (NRs) is a complex process requiring dissociation of corepressors and recruitment of coactivators. The putative transcriptional intermediary factor TIF1 alpha has been previously characterized as a nuclear protein that interacts directly with the AF-2 ligand-dependent activating domain present in the ligand-binding domain of numerous steroid and nonsteroid receptors, including the estrogen (ER alpha) and retinoid X (RXR alpha) receptors. We report here that TIF1 alpha is both a phosphoprotein and a protein kinase. TIF1 alpha coexpressed in COS-l cells with RXRa or ERa! is phosphorylated and becomes hyperphosphorylated upon ligand treatment. This hyperphosphorylation requires the binding of TIF1 alpha to transcriptionally active NRs since it is prevented by mutations either in the core (alpha-helix 12 of the ligand-binding domain) of the AF-2 activating domains of RXR alpha and ER alpha or in the NR box of TIF1 alpha that are known to prevent TIF1 alpha-NR interactions. Thus, TIF1 alpha is a phosphoprotein that undergoes ligand-dependent hyperphosphorylation as a consequence of nuclear receptor binding. We further show that purified recombinant TIF1 alpha possesses intrinsic kinase activity and that, in addition to autophosphorylation, TIF1 alpha selectively phosphorylates the transcription factors TFIIE alpha, TAF(II)28, and TAF(II)55 in vitro. These latter results raise the possibility that TIF1 alpha may act, at least in part, by phosphorylating and modifying the activity of components of the transcriptional machinery.