In response to IFN-γ, the latent cytoplasmic protein signal transducers and activators of transcription 1 (Stat1) becomes phosphorylated on Y701, dimerizes, and accumulates in the nucleus to activate transcription of IFN-γ-responsive genes. For maximal gene activation, S727 in the transcription activation domain of Stat1 also is inducibly phosphorylated by IFN-γ. We previously purified a group of nuclear proteins that interact specifically with the Stat1 transcription activation domain. In this report, we identified one of them as the multifunctional Ca 2+ /calmodulin-dependent kinase (CaMK) II. We demonstrate that IFN-γ mobilizes a Ca 2+ flux in cells and activates CaMKII. CaMKII can interact directly with Stat1 and phosphorylate Stat1 on S727 in vitro . Inhibition of Ca 2+ flux or CaMKII results in a lack of S727 phosphorylation and Stat1-dependent gene activation, suggesting in vivo phosphorylation of Stat1 S727 by CaMKII. Thus two different cellular signaling events, IFN-γ receptor occupation and Ca 2+ flux, are required for Stat1 to achieve maximal transcriptional activation through regulation of phosphorylation.

In response to IFN-␥, the latent cytoplasmic Stat1 (signal transducer and activator of transcription) proteins translocate into the nucleus and activate transcription. We showed previously that Stat1 recruits a group of nuclear proteins, among them MCM5 (minichromosome maintenance) and MCM3, for transcription activation. MCM5 directly interacts with the transcription activation domain (TAD) of Stat1 and enhances Stat1-mediated transcription activation. In this report, we identified two specific residues (R732, K734) in MCM5 that are required for the direct interaction between Stat1 and MCM5 both in vitro and in vivo. MCM5 containing mutations of R732͞K734 did not enhance Stat1-mediated transcription activation in response to IFN-␥. In addition, it also failed to form complexes with other MCM proteins in vivo, suggesting that these two residues may be important for an interaction domain in MCM5. Furthermore, MCM5 bearing mutations in its ATPase and helicase domains did not enhance Stat1 activity. In vitro binding assays indicate that MCM3 does not interact directly with Stat1, suggesting that the presence of MCM3 in the group of Stat1TAD-interacting proteins is due to the association of MCM3 with MCM5. Finally, gel filtration analyses of nuclear extracts from INF-␥-treated cells demonstrate that there is a MCM5͞3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5͞3 subcomplex through direct interaction with MCM5 in the process of IFN-␥-induced gene activation.

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