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DNA binding controls inactivation and nuclear accumulation of the transcription factor Stat1
Thomas Meyer1, Andreas Marg, Petra Lemke
1Abteilung Zelluläre Signalverarbeitung, Freie Universität Berlin, Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany.
Abstract:
Cytokine-dependent gene transcription greatly depends on the tyrosine phosphorylation ("activation") of Stat proteins at the cell membrane. This rapidly leads to their accumulation in the nucleus by an unknown mechanism. We performed microinjections of recombinant Stat1 protein to show that nuclear accumulation of phosphorylated Stat1 can occur without cytokine stimulation of cells. Microinjection of Stat1 antibody and treatment of cells with kinase or phosphatase inhibitors revealed that nuclear accumulation is a highly dynamic process sustained by Stat1 nucleocytoplasmic cycling and continuous kinase activity. By characterizing nuclear accumulation mutants, it is demonstrated that nuclear import and nuclear retention are two separate steps leading up to nuclear accumulation, with nonspecific DNA binding of activated Stat1 being sufficient for nuclear retention. Critical for nuclear buildup of Stat1 and the subsequent nuclear export is the point of time of tyrosine dephosphorylation, because our data indicate that activated Stat1 is incapable of leaving the nucleus and requires dephosphorylation to do so. It is demonstrated that the inactivation of Stat1 is controlled by its exchange reaction with DNA, whereby DNA binding protects Stat1 from dephosphorylation in a sequence-specific manner. Thus, during nuclear accumulation, a surprisingly simple mechanism integrates central aspects of cytokine-dependent gene regulation, for example, receptor monitoring, promoter occupancy, and transcription factor inactivation.
Insights
Signal transducer and activator of transcription (Stat) protein nuclear accumulation is a dynamic process involving continuous cycling and kinase activity. DNA binding protects activated Stat1 from dephosphorylation, controlling gene transcription regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Cytokine signaling relies on Signal transducer and activator of transcription (Stat) protein activation via tyrosine phosphorylation.
- Activated Stat proteins translocate to the nucleus to regulate gene transcription through an incompletely understood mechanism.
Purpose of the Study:
- To elucidate the mechanism of Stat1 nuclear accumulation independent of cytokine stimulation.
- To characterize the dynamic processes governing Stat1 nucleocytoplasmic transport and nuclear retention.
Main Methods:
- Microinjection of recombinant Stat1 protein and Stat1 antibodies into cells.
- Treatment with kinase and phosphatase inhibitors.
- Analysis of nuclear accumulation mutants and DNA-binding interactions.
Main Results:
- Nuclear accumulation of phosphorylated Stat1 can occur without cytokine stimulation.
- Stat1 nuclear accumulation is a dynamic process requiring continuous kinase activity and nucleocytoplasmic cycling.
- Nuclear import and retention are distinct steps; DNA binding mediates nuclear retention and protects Stat1 from dephosphorylation.
- Dephosphorylation is essential for Stat1 nuclear export, with DNA binding influencing inactivation rate.
Conclusions:
- A simple mechanism integrates cytokine-dependent gene regulation aspects, including receptor monitoring, promoter occupancy, and transcription factor inactivation.
- Stat1 inactivation is controlled by DNA exchange reactions, with sequence-specific DNA binding protecting Stat1 from dephosphorylation.