Phosphorylation of IRF-3 on Ser 339 Generates a Hyperactive Form of IRF-3 through Regulation of Dimerization and CBP Association
Bibliographic record
Abstract
The IkappaB kinase-related kinases, TBK1 and IKKi, were recently shown to be responsible for the C-terminal phosphorylation of IRF-3. However, the identity of the phosphoacceptor site(s) targeted by these two kinases remains unclear. Using a biological assay based on the IRF-3-mediated production of antiviral cytokines, we demonstrate here that all Ser/Thr clusters of IRF-3 are required for its optimal transactivation capacity. In vitro kinase assays using full-length His-IRF-3 as a substrate combined with mass spectrometry analysis revealed that serine 402 and serine 396 are directly targeted by TBK1. Analysis of Ser/Thr-to-Ala mutants revealed that the S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association, and nuclear accumulation. However, production of antiviral cytokines was still present in IRF-3 S396A-expressing cells. Interestingly, mutation of serine 339, which is involved in IRF-3 stability, also abrogated CBP association and dimerization without affecting gene transactivation as long as serine 396 remained available for phosphorylation. Complementation of IRF-3-knockout mouse embryonic fibroblasts also revealed a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce expression of the interferon-stimulated genes ISG56 and ISG54. These data lead us to reconsider the current model of IRF-3 activation. We propose that conventional biochemical assays used to measure IRF-3 activation are not sensitive enough to detect the small fraction of IRF-3 needed to elicit a biological response. Importantly, our study establishes a molecular link between the role of serine 339 in IRF-3 homodimerization, CBP association, and its destabilization.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".