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PreprintCited by 2Cytokine Signaling Pathways and InteractionsPsoriasis: Treatment and PathogenesisGenomics and Chromatin Dynamics

CDK8 fine-tunes IL-6 transcriptional activities by limiting STAT3 resident time at the gene loci

Open access
Martinez-Fabregas, J., Wang, L., Pohler, E., Cozzani, A., Kazemian, M., Mitra, S., Moraga, I.·2020·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.1101/2020.03.19.998351
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Abstract

Cytokines are highly pleiotropic ligands that critically contribute to a balanced immune response. We have an incomplete understanding of how cytokines elicit their functional pleiotropy, which has limited their therapeutic use. Here, using Interleukin-6 (IL-6) as a model system, we have performed detailed phosphoproteomic and transcriptomic studies in human Th-1 cells to address the molecular bases defining cytokine functional pleiotropy. We have identified CDK8 as a new negative regulator of STAT3 transcriptional activities that contributes to the diversification of IL-6 responses. We found that CDK8 is a major regulator of the IL-6 phosphoproteome and interacts with STAT3 in the nucleus upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecules inhibitors, reduced the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 Ser727 phosphorylation. This, in turn, resulted in retention of tyrosine phosphorylated STAT3 in the nucleus, which increased the binding of STAT3 to target DNA sites in the genome with a concomitant increase in STAT3 mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity via modulation of its gene loci resident time, critically contributing to tuning STAT3 mediated responses.

AI snapshot

Written from the paper’s full text.

TL;DR

Using IL-6 as a model cytokine in human primary Th-1 cells, this study identifies CDK8 (with an accessory role for CDK9) as a negative regulator of STAT3 transcriptional activity that phosphorylates STAT3 Ser727 in the nucleus and limits its chromatin resident time. Inhibiting CDK8 prolongs STAT3 chromatin binding, amplifies STAT3-dependent gene expression, and enhances Th-17 differentiation in vitro.

Key findings

  • IL-6 (delivered as Hyper-IL-6) preferentially activates JAK/STAT1/STAT3 in resting and activated human CD4+ and CD8+ T cells; activated T cells showed 2–3-fold higher STAT1/3 phosphorylation amplitudes than resting cells, and STAT3 Ser727 phosphorylation was delayed relative to Tyr705 and occurred after nuclear translocation.
  • Phosphoproteomics in Th-1 cells identified 17,935 phosphosites on 4,196 proteins, with 304 increased and 245 decreased by HyIL-6; nearly 40% of IL-6-induced phosphoproteomic changes were nuclear. A second SILAC experiment (11,035 phosphosites in 3,500 proteins) found 162 upregulated and 160 downregulated sites, of which 88 (63 up, 25 down) were CDK8-sensitive; CDK8 inhibition reduced the IL-6-induced phosphoproteome by 23%.
  • CDK8 inhibitors (CBI-2347, MSC2530818) reduced HyIL-6-induced STAT3 Ser727 phosphorylation by ~70%, whereas CDK9 or CDK12/13 inhibitors reduced it by only ~20%; none affected STAT3 Tyr705. PLA showed a 2–4-fold increase in nuclear STAT3/CDK8 and STAT3/CDK9 complexes peaking at 30 min, and CDK inhibition increased these complexes even without IL-6.
  • RNA-Seq: HyIL-6 alone dysregulated 27 genes, CDK8 inhibitor alone 111 genes (84 up), and combined treatment 176 genes; ChIP-Seq showed STAT3 binding intensity was synergistically increased with HyIL-6 + CDK8 inhibitor (2,585 shared bound regions vs 540 with HyIL-6 alone). CDK8 inhibition produced an average ~3-fold increase in IL-17-positive cells after 10 days under Th-17 polarizing conditions (baseline ~2–3% IL-17+), with increased secreted IL-17 but no change in RORγt, IFNγ, or IL-4.

Methods

  • Primary human CD4+ and CD8+ T cells from healthy donor buffy coats were stimulated with Hyper-IL-6 (HyIL-6); signaling was profiled by phospho-flow with a 28-marker antibody array, dose-response and kinetic experiments, and Western blotting of cytoplasmic/nuclear fractions.
  • SILAC-based quantitative phosphoproteomics (light/medium/heavy labeling, HILIC fractionation, Ti-IMAC enrichment, Q Exactive Plus MS, MaxQuant) in Th-1-polarized cells stimulated with HyIL-6 ± the CDK8 inhibitor MSC2530818; six biological replicates in the first experiment.
  • Proximity ligation assays for STAT3/CDK8 and STAT3/CDK9 complexes (including STAT3 WT-GFP vs Ser727Ala-GFP reconstitution in CRISPR STAT3-knockdown Hut78 cells), STAT3 ChIP-Seq with motif analysis (MEME/TOMTOM), RNA-Seq with GSEA, and in vitro human Th-17 differentiation assays with flow cytometry and ELISA.

Limitations

  • The paper states no dedicated limitations section. It notes that HyIL-6 was used to overcome variable IL-6 receptor expression across T cell populations, and that STAT3 overexpression in reconstituted Hut78 cells produced higher baseline STAT3/CDK9 complexes than in primary Th-1 cells.
  • The text makes evident that Torin1's inhibition of STAT1/STAT3 Ser phosphorylation is a previously unknown off-target effect (other mTOR inhibitors and ATM/DNA-PK inhibitors failed to reproduce it), and that in vitro Th-17 differentiation was inefficient (only ~2–3% IL-17-positive cells at baseline).
  • This is a bioRxiv preprint explicitly marked as not certified by peer review.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1101/2020.03.19.998351

Cites

106 of 106 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

19 of 34 cited sources checked · 9 supported · 0 flagged · accuracy not yet measured

  • Findingown data
    § These authors share senior authorship

    Flavopiridol completely abolished the serine phosphorylation of STAT3 induced by HyIL-6.

    “Flavopiridol, a well-described pan-CDK inhibitor (Luke et al., 2012), completely abolished the Ser phosphorylation of STAT3 induced by HyIL-6”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Luke et al., 2012 source
  • Premisecitation
    § These authors share senior authorship

    IL-6 receptor expression varies significantly among different T cell populations and environmental contexts.

    “IL-6 receptor expression varies significantly among different T cell populations and environmental contexts”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Betz and Muller, 1998 source
    • cites Jones et al., 2010 source
    • cites Ridgley et al., 2019 source

      The Results section reports significantly different surface IL-6R expression among naïve, central-memory, and effector-memory CD4+ T cells, with the highest expression on central-memory cells. The paper discusses how conditions affect IL-6 responses, but the passages do not show that environmental context changes receptor expression. Page numbers were not provided. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingown data
    § These authors share senior authorship

    Inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation.

    “Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation.”
    Quote in the paperSubstantial claim
  • Findingown data
    § These authors share senior authorship

    The majority of phosphopeptides modified by IL-6 treatment belonged to nuclear proteins.

    “This study revealed that the majority of phosphopeptides modified by IL-6 treatment belonged to nuclear proteins”
    Quote in the paperSubstantial claim
  • Findingreasoning
    § These authors share senior authorship

    Inhibition of CDK8 activity increased the binding of STAT3 to target DNA sites in the genome.

    “This, in turn, resulted in retention of tyrosine phosphorylated STAT3 in the nucleus, which increased the binding of STAT3 to target DNA sites in the genome with a concomitant increase in STAT3 mediated transcriptional activity.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 27
  • May combine two claims. 27
  • The quote only partly states this claim. 2

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § These authors share senior authorship

    CDKs are regulators of STAT1 serine phosphorylation in different systems.

    “Previous studies have described CDKs as regulators of STAT1 Ser phosphorylation in different systems”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Bancerek et al., 2013 source
    • cites Chen et al., 2019 source
    • cites Kosciuczuk et al., 2019

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    Torin 1 inhibited the serine phosphorylation induced by HyIL-6 in both STAT1 and STAT3, without affecting their tyrosine phosphorylation.

    “only Torin 1, an mTOR inhibitor targeting both mTORC1 and mTORC2 complexes (Thoreen et al., 2009), inhibited the Ser phosphorylation induced by HyIL-6 in both STAT1 and STAT3, without affecting their Tyr phosphorylation”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Thoreen et al., 2009 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § These authors share senior authorship

    In macrophages, CDK8 is the main kinase driving STAT1 serine phosphorylation in response to IFN gamma stimulation.

    “In macrophages, CDK8 appears to be the main kinase driving STAT1 serine phosphorylation in response to IFN gamma stimulation”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Bancerek et al., 2013 source

      The Results section ('CDK8 Phosphorylates S727') shows that CDK8 silencing reduced IFN-γ-induced STAT1 S727 phosphorylation more than CDK9 silencing, but those experiments were in fibroblasts. In bone-marrow-derived macrophages, the Results section ('A Nuclear CDK Mediates Canonical TAD Serine Phosphorylation') shows inhibition by flavopiridol, which is not specific to CDK8. The paper therefore supports CDK8 as a key kinase in this response, but does not establish that it is the main kinase specifically in macrophages. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § These authors share senior authorship

    HyIL-6 triggers signaling in all cells expressing gp130, producing a more robust and homogenous signaling output.

    “HyIL-6 triggers signaling in all cells expressing gp130, producing a more robust and homogenous signaling output”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Rose-John, 2012 source

      The Introduction (page not provided) says that gp130-expressing cells can be stimulated through IL-6 trans-signaling even without membrane IL-6R. The section “Protein tools to study IL-6 trans-signaling” reports that Hyper-IL-6 is 100–1,000 times more active than separate IL-6 and sIL-6R on gp130-expressing cells, supporting a more robust response. It does not establish that the signaling output is homogeneous across those cells. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § These authors share senior authorship

    Mutation of STAT3 Ser727 to Ala modulates STAT3 transcriptional activity.

    “Previous studies have reported that mutation of Ser727 in STAT3 to Ala modulates its transcriptional activity (Chung et al., 1997; Kim and Baumann, 1997; Lim and Cao, 1999; Wen et al., 1995; Yokogami et al., 2000).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Chung et al., 1997 source

      The Results section (page not available) reports that the STAT3 S727A mutation increased tyrosine phosphorylation, a step relevant to transcriptional activity. The Discussion proposes an effect on transcription but explicitly says future studies are needed to determine it; this paper does not establish that the mutation modulates transcriptional activity. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Kim and Baumann, 1997 source
    • cites Lim and Cao, 1999 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation
    § These authors share senior authorship

    TCR+IL-2 treatment of resting and activated human CD4+ and CD8+ T cells activated a vast array of signaling intermediaries detected by the antibody array, serving as a positive control.

    “In both resting and activated human CD4+ and CD8+ T cells, TCR+IL-2 treatment led to the activation of a vast array of signaling intermediaries, including STAT1, STAT3, STAT4, STAT5, STAT6, ERK, AKT, S6R, CREB”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Ross et al., 2016 source

      The Results section (‘The IL-2-Regulated Phosphoproteome’; page unavailable) reports that IL-2 stimulation changed many phosphorylations in CD8+ cytotoxic T cells, including STAT5 and ERK. It does not establish the claimed response to combined TCR+IL-2 treatment in resting and activated human CD4+ and CD8+ cells; the study used mass spectrometry rather than an antibody array and does not support the full list of intermediaries. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Smith-Garvin et al., 2009 source

      The DAG-Mediated Signaling Pathways section says TCR signaling activates ERK and STAT3, supporting part of the signaling claim. The review does not report TCR+IL-2 treatment of resting and activated human CD4+ and CD8+ cells, an antibody-array positive control, or activation of the full listed set of intermediaries. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § These authors share senior authorship

    STAT3 can engage different gene expression programs in a cell-dependent and stimulating condition-dependent manner.

    “several studies have reported that STAT3 can engage different gene expression programs in a cell-dependent and stimulating condition-dependent manner (Hutchins et al., 2013)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Hutchins et al., 2013 source

  • May combine two claims.

    Interpretationcitation
    § These authors share senior authorship

    STAT3 Ser phosphorylation plays a negative role in STAT3 nuclear dynamics and transcriptional activities.

    “our study and others (Chung et al., 1997; Decker and Kovarik, 2000; Kim and Baumann, 1997) highlight a negative role of STAT3 Ser phosphorylation in STAT3 nuclear dynamics and transcriptional activities.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Chung et al., 1997 source

      The Abstract and the section on STAT3 serine 727 phosphorylation report that serine phosphorylation negatively modulates tyrosine phosphorylation, which is required for nuclear translocation and subsequent transcriptional activity. But the Discussion says its effects on nuclear translocation and transcriptional activity still require study and proposes that serine phosphorylation can also stimulate transcription. The source therefore supports a possible negative effect, not the unqualified claim about both outcomes. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Decker and Kovarik, 2000 source
    • cites Kim and Baumann, 1997 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § These authors share senior authorship

    Small molecule inhibitors targeting CDK8 promote the differentiation of T regulatory cells and Th-17 cells under their respective polarizing conditions.

    “small molecule inhibitors targeting CDK8 promote the differentiation of T regulatory (reg) cells, and Th-17 cells when T cells were placed under T reg polarizing conditions (Akamatsu et al., 2019; Guo et al., 2019), or Th-17 polarizing conditions (Figure 8) respectively”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Akamatsu et al., 2019 source
    • cites Guo et al., 2019 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § These authors share senior authorship

    Ser727Ala mutation impairs transcriptional activity by different STATs.

    “several studies have shown that Ser727Ala mutation impairs transcriptional activity by different STATs (Levy and Darnell, 2002; Lim and Cao, 1999; Sadzak et al., 2008; Wen et al., 1995)”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Levy and Darnell, 2002 source
    • cites Lim and Cao, 1999 source
    • cites Sadzak et al., 2008 source

  • May combine two claims.

    Study design and conductown data
    § These authors share senior authorship

    CDK8 inhibition by MSC2530818 did not alter the phosphorylation profile of RBP1, indicating the treatment did not affect basal transcriptional programs.

    “CDK8 inhibition by MSC2530818 did not alter the phosphorylation profile of RBP1, suggesting that our treatment did not affect basal transcriptional programs (Sup. Figure 9B)”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Czodrowski et al., 2016 source

      The Supporting Information summary (p. 1) identifies MSC2530818 as a CDK8 inhibitor, and the compound-characterization passage (p. 8) reports cellular CDK8 activity. Neither passage reports an RBP1 phosphorylation profile or shows that basal transcriptional programs were unaffected; the citing sentence points to its own Supplementary Figure 9B for that observation. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites Harlen and Churchman, 2017 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § These authors share senior authorship

    Naïve T cells express non-detectable levels of CDK8, which are upregulated upon T cell activation and differentiation.

    “Naïve T cells express non-detectable levels of CDK8, which are upregulated upon T cells activation and differentiation (Howden et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Howden et al., 2019 source

      The Results section on cell-context effects of mTORC1 inhibition describes undetectable cyclin D3 and low CDK4/6 in naïve T cells, followed by increases after activation. Searches for CDK8 and cyclin-dependent kinase 8 found no passage in our copy stating that CDK8 is undetectable in naïve cells or upregulated with activation and differentiation. The available passages have no page numbers. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § These authors share senior authorship

    Annotating the shared STAT3 bound regions with the nearest gene yielded 475 unique genes.

    “The shared STAT3 bound regions were annotated with the nearest gene by 'annotatePeaks' from HOMER v4.10 (Gupta et al., 2007), yielding 475 unique genes.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Gupta et al., 2007 source

      The Abstract and Discussion describe Tomtom as a tool for comparing motifs, not for annotating genomic regions to nearby genes. Searches for STAT3, HOMER, annotatePeaks, nearest-gene annotation, and 475 unique genes found no passage addressing the claimed analysis. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    304 phosphosites were increased and 245 were decreased in response to HyIL-6 stimulation in human primary Th-1 cells.

    “Among those 304 were increased and 245 were decreased in response to HyIL-6 stimulation in human primary Th-1 cells”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    Inhibition of CDK activity using Flavopiridol produced a prolonged STAT3 tyrosine phosphorylation and the retention of tyrosine phosphorylated and total STAT3 in the nuclear fraction.

    “inhibition of CDKs activity using Flavopiridol produced a prolonged STAT3 Tyr phosphorylation and the retention of Tyr phosphorylated and total STAT3 in the nuclear fraction”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    Inhibition of CDK8 and CDK9 produced a more sustained STAT3 tyrosine phosphorylation and a longer STAT3 nuclear retention upon IL-6 stimulation.

    “inhibition of these kinases produced a more sustained STAT3 tyrosine phosphorylation and a longer STAT3 nuclear retention upon IL-6 stimulation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    STAT3 tyrosine phosphorylation is fast and takes place in the cytoplasm, while STAT3 serine phosphorylation is delayed and occurs after STAT3 nuclear translocation.

    “nuclear fractionation studies revealed that while STAT3 Tyr phosphorylation is fast and takes place in the cytoplasm, STAT3 Ser phosphorylation is delayed and occurs after STAT3 nuclear translocation”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    Upon HyIL-6 stimulation, a 2-4 fold increase in the number of STAT3/CDK8 and STAT3/CDK9 complexes was detected, which peak at 30 min stimulation and return to basal levels after 3 hours.

    “Upon HyIL-6 stimulation, we detected a 2-4 fold increase in the number of STAT3/CDK8 and STAT3/CDK9 complexes, which peak at 30 min stimulation and return to basal levels after 3 hours”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § These authors share senior authorship

    The phosphoproteomic study identified 288 kinases.

    “Our phosphoproteomic study identified 288 kinases (Figure 2D and Sup. Table S1). Of those 28 were regulated by HyIL-6 treatment”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § These authors share senior authorship

    GO analysis showed an enrichment of the JAK/STAT pathway upon stimulation of Th-1 cells with HyIL-6.

    “GO analysis (KEGG pathways and molecular function analyses) showed an enrichment of the JAK/STAT pathway as expected upon stimulation of Th-1 cells with HyIL-6”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    HyIL-6 treatment induced upregulation of 162 and downregulation of 160 phosphosites, of which 88 (63 upregulated and 25 downregulated) were sensitive to CDK8 inhibition.

    “HyIL-6 treatment induced the upregulation of 162 and downregulation of 160 phosphosites, where 88 of them (63 of the upregulated and 25 of the downregulated) were sensitive to CDK8 inhibition”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § These authors share senior authorship

    The phosphoproteomics study identified 11,035 phosphosites in 3,500 proteins.

    “The combined analysis of our phosphoproteomics study showed the identification of 11,035 phosphosites in 3,500 proteins (Figure 6A and 6B and Sup. table S2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data
    § These authors share senior authorship

    In STAT3-reconstituted Hut78 cells, unstimulated cells had significantly higher levels of STAT3/CDK9 complex than human Th-1 cells, due to STAT3 over-expression.

    “Due to STAT3 over-expression in these cells, we detected significantly higher levels of STAT3/CDK9 complex in unstimulated cells than those detected in human Th-1 cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    27% of proteins involved in transcription and 40% of proteins involved in RNA Pol II transcription were affected by CDK8 inhibition.

    “27% of proteins involved in transcription and 40% of proteins involved in RNA Pol II transcription were affected by CDK8 inhibition (Figure 6F).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data
    § These authors share senior authorship

    These non-nuclear phosphosites were also enriched in Ser/Thr phosphorylation.

    “These phosphosites were also enriched in Ser/Thr phosphorylation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    In unstimulated Th-1 cells, STAT3 DNA binding was very poor and was significantly enhanced upon HyIL-6 treatment.

    “in unstimulated Th-1 cells, we detected very poor STAT3 DNA binding, which was significantly enhanced upon HyIL-6 treatment (Figure 7E)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    CDK8 inhibition increased secreted IL-17 levels as measured by ELISA, but not IFNg.

    “CDK8 inhibition resulted in an increase in the levels of secreted IL-17 as measured by ELISA (Figure 8J), but not IFNg (Figure 8K).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    STAT3 Ser727A mutant exhibited a similar nuclear translocation profile to STAT3 wt but a delayed association kinetic with CDK9.

    “STAT3 Ser727A mutant exhibited a similar nuclear translocation profile to STAT3 wt, but it showed a delayed association kinetic with CDK9 (Figure 5E).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The STAT3 Ser727Ala mutant is not phosphorylated at Ser727 and faithfully mimics the absence of STAT3 Ser727 phosphorylation.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation
    § These authors share senior authorship

    HyIL-6 treatment induced a minor increase in the number of human Th-17 cells.

    “HyIL-6 treatment induced a minor increase in the number of human Th-17 cells, highlighting the difficulty of working with these cells in vitro”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § These authors share senior authorship

    Upon HyIL-6 stimulation, the number of STAT3 wt/CDK9 complexes in the nucleus significantly increased, peaked at 30 min, and returned to basal levels by two hours of treatment.

    “upon HyIL-6 stimulation we observed a significant increase in the number of STAT3 wt/CDK9 complexes in the nucleus, which peaked at 30 min and went back to basal levels by two hours treatment (Figure 5E).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data
    § These authors share senior authorship

    HyIL6 stimulation induces phosphosites that are downregulated in a CDK8-dependent manner.

    “CDK8-dependent HyIL6 downregulated phosphosites”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § These authors share senior authorship

    There are 475 STAT3 binding genes.

    “STAT3 Binding genes (N=475)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § These authors share senior authorship

    Blockage of STAT1 serine phosphorylation significantly altered the IFN gamma transcriptional response and its ability to clear Listeria monocytogenes infection.

    “Blockage of STAT1 serine phosphorylation, using STAT1 serine mutants, significantly altered the IFN gamma transcriptional response and its ability to clear Listeria monocytogenes infection”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Varinou et al., 2003 source

  • May combine two claims.

    Interpretationown data
    § These authors share senior authorship

    CDK8 plays a critical role in regulating STAT3 serine phosphorylation by HyIL-6, with an accessory role of other CDK members.

    “These data highlight a critical role of CDK8 in regulating STAT3 Ser phosphorylation by HyIL-6, with an accessory role of other CDK members.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § These authors share senior authorship

    CDK8 is a new negative regulator of STAT3 transcriptional activities that contributes to the diversification of IL-6 responses.

    “We have identified CDK8 as a new negative regulator of STAT3 transcriptional activities that contributes to the diversification of IL-6 responses.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § These authors share senior authorship

    SILAC labelling incorporation greater than 97% was confirmed by mass spectrometry.

    “Labelling incorporation greater than 97% was confirmed by Mass Spectrometry.”
    • Also stated in: § These authors share senior authorship · plain
      “Labelling incorporation greater than 97% was confirmed by Mass Spectrometry.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § These authors share senior authorship

    IL-6 strongly regulates the nuclear phosphoproteome, which could contribute to fine-tune its immuno-modulatory activities.

    “Overall our phosphoproteomic revealed a strong regulation of the nuclear phosphoproteome by IL-6 that could contribute to fine-tune its immuno-modulatory activities.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § These authors share senior authorship

    STAT1 Ser727Ala knock-in mice elicited a poor immune response against Listeria monocytogenes infection.

    “STAT1 Ser727Ala Knock-in mice elicited a poor immune response against Listeria monocytogenes infection (Varinou et al., 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Varinou et al., 2003 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § These authors share senior authorship

    The quality of the sequencing libraries was inspected using FastQC v0.11.8.

    “The quality of libraries was inspected using FastQC v0.11.8.”
    • Also stated in: § These authors share senior authorship · plain
      “The quality of libraries was inspected by FastQC v0.11.8.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation
    § These authors share senior authorship

    CDK8 Ser phosphorylates STAT3, triggering its dissociation from chromatin and terminating STAT3-mediated transcription.

    “CDK8 Ser phosphorylates STAT3 triggering its dissociation from chromatin and terminating STAT3-mediated transcription.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § These authors share senior authorship

    CD4+ T cells were isolated by MACS to a purity of at least 99%.

    “isolated by magnetic activated cell sorting (MACS, Miltenyi) using anti-FiTC microbeads (Miltenyi, #130-048-701) according to manufacturer's instructions to a purity ≥99%.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § These authors share senior authorship

    CDK8 is recruited to STAT3 binding gene loci upon IL-6 stimulation, where it triggers STAT3 Ser phosphorylation and limits STAT3 chromatin resident time.

    “CDK8 is recruited to STAT3 binding gene loci upon IL-6 stimulation, where it triggers STAT3 Ser phosphorylation, and limits STAT3 chromatin resident time.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation
    § These authors share senior authorship

    p-values were determined by Student's t test and corrected for multiple testing using the Benjamini-Hochberg procedure.

    “the p-values were determined by Student´s t test and corrected for multiple testing using the Benjamini–Hochberg procedure (Benjamini and Hochberg, 1995).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Benjamini and Hochberg, 1995 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § These authors share senior authorship

    Recombinant HyIL-6 was purified to greater than 98% homogeneity.

    “Recombinant HyIL-6 was purified to greater than 98% homogeneity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § These authors share senior authorship

    CDK8 modulates STAT3 transcriptional processivity by fine-tuning its gene loci binding dwell-time, leading to negative regulation of IL-6-mediated activities.

    “Overall, our data agree with a model where CDK8 modulates STAT3 transcriptional processivity by fine-tuning its gene loci binding dwell-time, leading to a negative regulation of IL-6-mediated activities.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § These authors share senior authorship

    Specific deletion of CDK8 in NK cells results in enhancement of their anti-tumor responses.

    “Specific deletion of CDK8 in NK cells results in an enhancement of their anti-tumor responses (Witalisz-Siepracka et al., 2018)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Witalisz-Siepracka et al., 2018 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § These authors share senior authorship

    Peptide and protein ratios were only considered when quantified in at least two out of the three replicates.

    “Peptide and protein ratios only quantified in at least two out of the three replicates were considered”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § These authors share senior authorship

    Identified ChIP-seq peaks were screened against hg19 blacklisted genomic regions, mitochondrial DNA, and pseudo-chromosomes.

    “The identified peaks were further screened against 'hg19 blacklisted' genomic regions, mitochondrial DNA, and pseudo-chromosomes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § These authors share senior authorship

    Statistical analyses were performed using two-tailed parametric and non-parametric tests as appropriate.

    “Statistical analyses were performed using the Two-tailed parametric and non-parametric tests as appropriate.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § These authors share senior authorship

    STAT3 binds CDK8 and CDK9 in the nucleus, where these two kinases drive its Ser phosphorylation.

    “Our data showed that STAT3 binds CDK8 and CDK9 in the nucleus, where these two kinases drive its Ser phosphorylation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § These authors share senior authorship

    A set of 199 STAT3-upregulated genes is available from GSE21670.

    “STAT3 up-regulated genes (N=199; GSE21670)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites GSE21670

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

51 of 51

  • The paper relies on this without support.

    The molecular bases governing the large functional pleiotropy of cytokines remain poorly defined.

    “the molecular bases governing their large functional pleiotropy remains poorly defined”

  • The paper relies on this without support.

    The molecular bases that allow IL-6 to elicit its vast array of immune-modulatory activities are not known.

    “The molecular bases that allow IL-6 to elicit its vast array of immune-modulatory activities are not known.”

  • The paper relies on this without support.

    The signaling networks engaged by IL-6 in T cells, and their role in shaping IL-6 immune activities, are poorly understood.

    “we have a poor understanding of the signaling networks engaged by IL-6 in T cells, and their role in shaping IL-6 immune activities”

  • The paper relies on this without support.

    The kinase responsible for STAT1/3 serine phosphorylation in T cells is not known.

    “the kinase responsible for STAT1/3 serine phosphorylation in T cells is currently not known”

  • The paper relies on this without support.

    The STAT3 Ser727Ala mutant is not phosphorylated at Ser727 and faithfully mimics the absence of STAT3 Ser727 phosphorylation.

    “STAT3 KnD cells were reconstituted with wt STAT3-GFP or Ser727A STAT3-GFP mutant and the levels of STAT3/CDK9 complex formation were measured by PLA (Figure 5E and Sup. Figure 7F).”
5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 1 legend gives the wrong STAT3 tyrosine site

    Minor

    The Figure 1 legend labels the STAT3 measurement as Tyr701, while the paper identifies the measured STAT3 site as Tyr705.

1 of 1

Cites

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Related

Authors
Martinez-Fabregas, J., Wang, L., Pohler, E., Cozzani, A., Kazemian, M., Mitra, S., & Moraga, I.
Affiliations
  • Purdue University West Lafayette (US)
  • Inserm (FR)
  • University of Dundee (GB)
Corresponding author
  • Kazemian, M.
  • Mitra, S.
  • Moraga, I.
ORCID
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2020-03-20
DOI
10.1101/2020.03.19.998351
OpenAlex
W3010843802
Semantic Scholar
53c0e67e80c6d6141880cfaed7dcd89fd8c632eb
Cited by
2
References
102
Citations by year
2021: 1 · 2019: 1
Type
posted content
Language
en
Open access
Licence
Locations
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • LillOA (Université de Lille (University Of Lille)) (submittedVersion, other-oa)
Topics
  • Health Sciences › Medicine › Oncology › Cytokine Signaling Pathways and Interactions
  • Life Sciences › Immunology and Microbiology › Immunology › Psoriasis: Treatment and Pathogenesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
CDK8, STAT3, interleukin-6, STAT3 phosphorylation, CDK8 inhibitors, phosphoproteome

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

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