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Immunology and Cell Biology (2014) 92, 737–738; doi:10.1038/icb.2014.71; published online 12 August 2014

Crosstalk between STATs and TLRs

TRAF6 is a nexus for TLR-STAT1 crosstalk

Jelena S Bezbradica1 and Kate Schroder1

1Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland, Australia

Correspondence: Kate Schroder, E-mail: K.Schroder@imb.uq.edu.au

Cells communicate with their extracellular environment via cell surface receptors. Cytokine receptors are a class of transmembrane receptors that recognize cognate cytokines (for example, interferons (IFNs)) released by other cells and transmit this information into the cell via signal transduction circuits involving receptor-associated Janus Kinase (JAK) family members and transcription factor(s) belonging to the signal transducer and activator of transcription (STAT) family.1 Despite their relatively simple signaling design, the JAK–STAT signal transduction module is not isolated; it communicates extensively with, and is modified by, other signaling modules that also transmit specific information about the extracellular environment. One such example is STAT communication with the family of pathogen-sensing Toll-like receptors (TLRs). In this issue, Luu et al.2 provide a mechanism for this signal crosstalk, implicating TLR-activated TRAF6 in facilitating serine phosphorylation of STAT1 in macrophages. The authors also describe a role for this STAT1 modification in TLR-mediated inflammation.

Two major cell signaling pathways regulate the activity of STATs (Figure 1): the canonical pathway elicited by cytokine receptors and JAKs,1 and the non-canonical pathway triggered by cellular stress (UV), TLRs, interleukin-1 receptor, tumor necrosis factor receptor, B- and T-cell receptors, and Fc-receptor for immunoglobulins (FcR).3 In the canonical pathway, cytokine–receptor interaction results in transphosphorylation of the two receptor-associated JAKs and cytokine receptor phosphorylation. These phosphorylation events create docking sites for STAT binding. Upon docking, JAKs phosphorylate STATs on a conserved tyrosine (Y) residue to allow their dissociation from the receptor, dimerization via reciprocal phospho-Y–SH2 STAT interactions, migration into the nucleus and gene regulatory function. Upon nuclear entry and positioning on target gene promoters, some STATs (STAT1/3/4/5a/5b) are further modified on a conserved serine (S) residue within their C-terminal transactivation domain by the nuclear serine kinase, CDK8.4,5 Serine phosphorylation of the transactivation domain critically regulates STAT transactivator function, as it allows the recruitment of transcription coactivators such as CBP/p300 to specific STAT-bound gene promoters, to regulate STAT-mediated transcription in vitro and in vivo.6,7 In contrast, in the non-canonical pathway, activation of STATs is independent of cytokine receptors, JAKs, STAT Y-phosphorylation and nuclear entry, and CDK82–5 (Figure 1). In fact, in the non-canonical pathway, a variety of receptors (for example, TLRs) trigger rapid STAT S- but not Y-phosphorylation directly in the cytosol, via poorly characterized pathways. The precise mechanisms by which STATs are targeted by non-canonical pathways and whether they are direct or indirect signaling substrates of these pathways have remained open questions. The biological functions of non-canonical STAT activation are also poorly characterized. STATs S-phosphorylated via the non-canonical pathway remain in the cytosol in a permissive state, enabling Y-phosphorylation through the canonical pathway should the cell subsequently encounter a cytokine signal. Hence, it is thought that one function of the non-canonical pathway may be to ‘prime’ cells to subsequent cytokine signals.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The canonical and non-canonical pathways for STAT activation. The canonical pathway (right) is elicited upon cytokine–receptor interaction. It involves activation of receptor-associated JAKs, JAK-mediated Y-phosphorylation of STATs followed by STAT dimerization and migration into the nucleus to initiate specific gene transcription. In the nucleus, some STATs can be additionally phosphorylated by CDK8 on the conserved S-residue in the C-terminal transactivation domain. Serine (S) phosphorylation critically regulates STAT transactivator function as it allows the recruitment of transcriptional coactivators (for example, CBP/p300) to specific STAT-bound gene promoters. The non-canonical pathway (left) is independent of cytokines, JAKs and STAT Y-phosphorylation, and nuclear translocation. In this pathway, STATs are phosphorylated on the same conserved S-residue within the transactivation domain in response to cellular stress (UV), or engagement of the TLRs, interleukin-1 receptor, tumor necrosis factor receptor, B- and T-cell receptors or Fc-receptor for immunoglobulins (FcR). The mechanism underlying the non-canonical pathway is still poorly understood, but involves S-phosphorylation of STAT in the cytosol. For example, in response to TLRs, TRAF6 directly recruits STAT1 into the TLR signalosome for S-phosphorylation. In earlier studies using pharmacological inhibitors, the MAPK family was implicated in the non-canonical pathway, but the identity of the cytosolic S-kinase for STATs still remains a matter of debate. The non-canonical pathway does not directly elicit STAT Y-phosphorylation, but STAT remains responsive to Y-phosphorylation should the canonical pathway be triggered by subsequent cytokine stimulation (e.g. TLR-induced type I IFN signaling). Hence, one function of the non-canonical pathway may be to ‘prime’ cells to subsequent cytokine exposure.

Full figure and legend (90K)

Luu et al.2 address the mechanism of non-canonical STAT activation using macrophage TLR signaling as a model system. TLRs sense conserved pathogen-associated molecular patterns and transmit these signals via MyD88/TRIF adapters, downstream interleukin-1 receptor-associated kinase 1 (IRAK) family kinases and the TRAF6 ubiquitin ligase.8 At this node, the pathway branches to activate nuclear factor-κB and mitogen-activated protein kinase cascades, and combinatorially orchestrate antimicrobial responses. Previous studies have documented STAT1 S727 phosphorylation downstream of TLR2/4/9 activation.9,10 However, it was unclear how TLRs target STAT1, and whether STAT1 modification was a common feature of all TLR signaling pathways. In a comprehensive screen, Luu et al.2 demonstrate that all TLRs, including plasma membrane (TLR2 and TLR4) and endosomal (TLR3, TLR7 and TLR9) receptors, can induce STAT1 S-phosphorylation. Of these, TLR2/4/7/9 appear to be able to do so directly, within minutes of their engagement, while TLR3-dependent STAT1 S-phosphorylation is delayed. Using cells from mice deficient in key TLR-associated adapter proteins Luu et al.2 demonstrate that STAT1 S-phosphorylation depended upon MyD88/TRIF, and involved direct interaction between STAT1 and TLR-activated TRAF6. These data are consistent with a previous report implicating IRAK1—the TLR-activated kinase upstream of TRAF6—in non-canonical STAT1 S727 phosphorylation.11 Thus, TRAF6 is an important signaling node that relays TLR signals to nuclear factor-κB, mitogen-activated protein kinase and STAT pathways to directly modulate key cellular processes.

Autocrine IFN signaling is an important component of the TLR pathway for many, but not all, TLRs. Using type I IFN receptor- or IFR3/7-deficient mice Luu et al.2 demonstrated that rapid non-canonical STAT1 S727 phosphorylation is direct and independent of autocrine type-I IFN signaling for all TLRs except TLR3. TLRs capable of inducing type I IFN (TLR3/4 in macrophages and TLR3/4/9 in dendritic cells) could additionally activate classical Y-phosphorylation of STAT1 via autocrine type I IFN, with somewhat delayed kinetics. Hence, these TLRs could drive STAT nuclear translocation via the type I IFN-directed canonical pathway. The second group of TLRs, which do not elicit type I IFN (TLR2/7 and TLR9 in macrophages) were only capable of directly activating S- but not Y-phosphorylation of STAT1, and hence did not promote nuclear translocation.10 Thus, Luu et al.2 describe direct crosstalk between TLR and STAT pathways, whereby TRAF6 recruits STAT1 into the TLR signalosome to facilitate STAT1 S-phosphorylation.

The study by Luu et al.2 provides important mechanistic insight into how STAT pathways are modified by TLRs. It also raises several questions: (a) Which kinase is recruited to the TLR signalosome to S-phosphorylate TRAF6-associated STAT1? (b) Do the other receptors that trigger the non-canonical pathway utilize shared or analogous mechanisms for non-canonical STAT1 activation? (c) What is the precise biological function of non-canonical STAT1 activation? As STAT1 S-phosphorylated by the non-canonical pathway can still serve as a substrate for canonical cytokine-induced STAT1 Y-phosphorylation, it is likely that the non-canonical pathway primes STAT1 to potentiate subsequent cytokine responses. It remains an open and interesting question whether STAT1-regulated genes primed by the two distinct pathways drive differing cellular programs. (d) As STAT1, which is S-phosphorylated by the non-canonical pathway but not Y-phosphorylated, is retained in the cytosol, what is the molecular function of S-phosphorylated STAT1 in the cytosol?

In summary, the STAT pathway can directly communicate with heterologous signaling pathways to integrate multiple extracellular cues and thereby elicit diverse cellular functions. How such mechanisms of signal crosstalk are translated into appropriate functional responses remains an important outstanding question.

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