The transcription factor Stat1 plays an essential role in responses to interferons (IFNs). Activation of Stat1 is achieved by phosphorylation on Y701 that is followed by nuclear accumulation. For full transcriptional activity and biological function Stat1 must also be phosphorylated on S727. The molecular mechanisms underlying the IFN-induced S727 phosphorylation are incompletely understood. Here, we show that both Stat1 Y701 phosphorylation and nuclear translocation are required for IFN-induced S727 phosphorylation. We further show that Stat1 mutants lacking the ability to stably associate with chromatin are poorly serine-phosphorylated in response to IFN-␥. The S727 phosphorylation of these mutants is restored on IFN-␤ treatment that induces the formation of the ISGF3 complex (Stat1/Stat2/ Irf9) where Irf9 represents the main DNA binding subunit. These findings indicate that Stat1 needs to be assembled into chromatinassociated transcriptional complexes to become S727-phosphorylated and fully biologically active in response to IFNs. This control mechanism, which may be used by other Stat proteins as well, restricts the final activation step to the chromatin-tethered transcription factor.kinase ͉ transcription T he Stat (signal transducers and activators of transcription) proteins are major cytokine-activated transcription factors that play a vital role in the biology of the hematopoietic and immune systems (1). Triggering of the cytokine receptor causes Stat tyrosine phosphorylation by the receptor-associated Jak tyrosine kinases causing the Stat homo-or heterodimers to accumulate in the nucleus and bind DNA. In addition, several Stat proteins are serine-phosphorylated in the C-terminal transactivation domain. Generation of knockin mice bearing alanine instead of serine at position 727, the site of Stat1 and Stat3 serine phosphorylation, and in vivo reconstitution experiments using Stat4 mutated at the phosphorylation site S721 proved the importance of these modifications for the transcriptional activity and biological function (2-4). Stat1 is activated in response to type I and type II interferons (IFNs) by phosphorylation at both Y701 and S727. On type I IFN (IFN-␣ and IFN-␤) stimulation Stat1 is assembled in the ISGF3 complex (Stat1, Stat2, and Irf9 heterotrimer) and, to a lesser extent, in Stat1 homodimers. The type II IFN-␥ activates primarily Stat1 homodimers. Several studies revealed that Stat1 complexes are, to a considerable part, preassembled before IFN stimulation (5-7). Phosphorylation of Y701 triggers Stat1 to accumulate in the nucleus in an importin-␣5-dependent manner (8, 9). Residues within the Stat1 N terminus as well as in the DNA binding domain were shown to be critical for the IFN-induced nuclear translocation (10-13). Stat1 can also shuttle between the cytoplasm and nucleus independently of IFN stimulation and Y701 phosphorylation (11). Both types of IFNs require S727-phosphorylated Stat1 for biological responses (14-16). The identity of the IFN-induced S727 kinase is not fully resolved. Th...

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