A rapid conformational rearrangement of STAT1 dimers is required for termination rather than for amplification of interferon-γ signaling

Abstract

Sequence-specific binding of STAT1 (signal transducer and activator of transcription 1) transcription factor to palindromic promoter elements, termed γ-activated sites (GAS), and an extended spatial reorientation between two dimer configurations are key events in the interferon signaling pathway. Although the DNA-binding domain of STAT1 is engaged in both processes, how the conformational change from a parallel to an antiparallel dimer configuration affects cytokine-induced target gene activation is unknown. In order to study the impact of the conformational shift on gene expression, we generated a STAT1 point mutant with a structurally altered architecture of the DNA-binding domain and characterized the resulting mutant (F364A) in cells stimulated with interferon-γ. Here, we report that substituting alanine for phenylalanine at position 364 resulted in reduced affinity to GAS sites and, additionally, a decreased dephosphorylation rate by the inactivating Tc45 phosphatase. The mutant had no defect in cooperative DNA binding and displayed normal kinetics of interferon-γ-induced nuclear accumulation, despite its elevated level of tyrosine phosphorylation. By assessing the transcriptional activity of the mutant, we found a strikingly robust expression of known interferon-γ-driven target genes, indicating that an impaired stability of the antiparallel dimer configuration can compensate for a reduced affinity to GAS sites. However, the mutant followed changes in ligand-induced receptor activation more slowly than the wild-type molecule, as demonstrated by its elevated phospho-STAT1 concentration following addition of the kinase inhibitor staurosporine to interferon-pretreated cells. This finding showed that the DNA-binding mutant F364A had partially lost its ability to terminate signal transmission rapidly. Thus, the coupling of high-affinity GAS binding to a rapid exchange from a parallel to an antiparallel dimer conformation is not necessarily required for optimal signal amplification, but rather allows for a dynamic signal response and ensures high adaptability to changes in signal input.

Keywords: :

• STAT1
• tyrosine phosphorylation
• DNA binding
• transcriptional regulation
• interferon signaling
• gene expression

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors gratefully acknowledge the excellent technical assistance of Anke Gregus and Heike Hühn. We kindly thank Dr Uwe Vinkemeier, University of Nottingham, for valuable reagents and discussions. The research on this subject was funded by a grant from the Deutsche Forschungsgemeinschaft to T.M.