Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-gamma.
In response to IFN-gamma, 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 R732/K734 did not enhance Stat1-mediated transcription activation in response to IFN-gamma. 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-gamma-treated cells demonstrate that there is a MCM5/3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5/3 subcomplex through direct interaction with MCM5 in the process of IFN-gamma-induced gene activation.
Department of Pathology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
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CJ
DaFonseca
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Shu
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JJ
Zhang
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D009687:Nuclear Proteins; D050794:STAT1 Transcription Factor; D029702:Schizosaccharomyces pombe Proteins; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C066470:MCM3 protein, human; D009687:Nuclear Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D029702:Schizosaccharomyces pombe Proteins; D015534:Trans-Activators; C085711:mcm5 protein, S pombe; D007371:Interferon-gamma; C099243:MCM4 protein, human; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4