It is commonly assumed that activation of a protein kinase is synonymous to phosphorylation of its substrates. This assumption has been applied to many kinase assays that rely on detection of activated kinases with phospho-specific antibodies or with surrogate substrates rather than physiological substrates. However, accumulating evidence suggests that activation of a kinase does not equate to phosphorylation of a physiological substrate. Recent studies of TBK1 provide a striking example of the uncoupling of protein kinase activation and substrate phosphorylation (30
). It is now well established that TBK1 is responsible for IRF3 phosphorylation in response to stimulation of several intracellular receptors that induce type-I interferons, including TLR3, TLR4, RIG-I, MDA5 as well cytosolic DNA sensors that signal through STING. IRF3 is not activated in inflammatory pathways triggered by other TLRs, such as TLR2 and TLR5, or by receptors for TNF or IL-1, all of which are known to activate NF-κB. However, TLR ligands and IL-1β could activate TBK1, raising the question of why activated TBK1 does not lead to IRF3 phosphorylation (30
). The answer to this question may lie in the fact that signaling pathways that lead to IRF3 activation engage specific adaptor proteins, such as TRIF for TLR3 and TLR4, MAVS for RIG-I and MDA5, and STING for the DNA sensing pathways.
Through in vitro reconstitution of STING-dependent IRF3 activation, we obtained evidence that may explain why IRF3 is activated only in a subset of pathways that stimulate TBK1. We found that STING binds to both TBK1 and IRF3 and that this binding is mediated through a short C-terminal fragment of STING (). Importantly, we identified two residues, Ser366 and Leu374, at the C-terminus of STING that are dispensable for TBK1 binding and activation, but required for IRF3 binding and phosphorylation. These results indicate that STING not only mediates TBK1 activation, but also specifies IRF3 for phosphorylation by TBK1. Although IL-1 and TNF can also stimulate TBK1, these ligands do not activate STING, hence no IRF3 phosphorylation. It will be very interesting to determine whether other adaptors such as MAVS and TRIF also activate IRF3 through the dual mechanism of stimulating TBK1’s catalytic activity and specifying IRF3 for phosphorylation by TBK1.
Burdette et al recently showed that STING binds directly to cyclic-di-GMP, which is known to induce type-I interferons (32
). A mouse STING mutant containing three substitutions at the C-terminus, S357A, E359A and S365A (equivalent to human STING S358A, E360A, S366A), retained less than 10% of IFNβ inducing activity of the wild type protein when it is overexpressed. Further, ectopic expression of this mutant in HEK293 cells did not confer IFNβ induction in response to cyclic-di-GMP. By knocking down endogenous STING in L929 cells and replacing it with human STING, we demonstrated that a single S366A mutation abolished its ability to bind and activate IRF3 following ISD stimulation ().
In our in vitro reconstitution system, only three proteins - TBK1, IRF3 and a C-terminal fragment of STING - are necessary and sufficient for STING-dependent IRF3 phosphorylation. This simple system is in contrast to MAVS-dependent phosphorylation of IRF3, which requires K63 polyubiquitination as well as detection of polyubiquitin chains by NEMO (28
). Consistent with this disparity, NEMO is required for IRF3 activation by MAVS, but not STING, in cells (fig. S3
). Unlike MAVS, which contains several binding sites for TRAF proteins, including TRAF2, TRAF3 and TRAF6, STING contains multiple transmembrane domains but no apparent TRAF binding motifs. Thus, STING activates TBK1 and promotes IRF3 phosphorylation through a rather simple mechanism, namely by direct binding to TBK1 and IRF3. However, our results do not mean that ubiquitination is not involved in the cytosolic DNA signaling pathway. In fact, it has been reported that TRIM56 ubiquitinates STING to modulate its ability to induce immune responses against intracellular DNA(33
). Therefore, it is possible that ubiquitination may control a step upstream of or at the level of STING activation.
The mechanism of STING activation by cytosolic DNA remains to be elucidated. Several DNA sensors have been proposed, including DAI, RNA polymerase III and IFI-16 and DDX41(16
). Further genetic studies are required to further establish the role of these and other potential DNA sensors in type-I interferon induction (12
). In any case, it has been demonstrated that STING is indispensable for interferon induction by cytosolic DNA in many cell types including macrophages (21
). Interestingly, microscopic studies have shown that STING forms punctate-like cytoplasmic structures. Previous studies have demonstrated that the TM domains of STING are essential for it to activate IRF3 and induce IFN (17
). Indeed, it has been shown that DDX41 interacts with STING in the region spanning the second to fourth transmembrane domains of STING (16
). Surprisingly, we found that the C-terminal fragment of STING containing just 39 amino acids is sufficient to support IRF3 phosphorylation by TBK1 in vitro. Interestingly, a fraction of the recombinant STING fragment eluted from the gel filtration column as high molecular weight species, and only these species were capable of promoting IRF3 phosphorylation by TBK1. Native gel electrophoresis also suggests that endogenous STING forms aggregates following ISD stimulation. Thus, it is possible that the detection of cytosolic DNA leads to aggregation and activation of STING on ER or other membranes, and that the TM domains of STING are required for its aggregation. Since a fraction of the recombinant STING fragment already forms active aggregates, the TM domains are no longer required. This mechanism is analogous to that of the activation of MAVS, which forms very large aggregates in response to RNA virus infection (34
). The mitochondrial TM domain of MAVS is required for it to form these functional aggregates in cells in response to viral infection. In the in vitro experiments, however, MAVS lacking the TM domain is active because a fraction of the recombinant protein already forms high molecular weight aggregates. The MAVS aggregates are highly potent in activating the IRF3 signaling cascade through a prion-like mechanism (34
). Thus far, we have not obtained any evidence that STING forms similar prion-like aggregates in the cytosolic DNA signaling pathway.
Another potential mechanism of STING regulation is suggested by our observation that STING is phosphorylated in response to stimulation by cytosolic DNA. This phosphorylation of STING depends on TBK1. Previous studies have shown that STING is phosphorylated at Ser358
by TBK1 in response to Sendai virus infection (19
). Our mass spectrometry analysis confirmed the phosphorylation of STING at Ser358
following ISD stimulation. However, the mutation of Ser358
to alanine only partially impaired the ability of STING to rescue IRF3 activation in STING-deficient cells. In contrast, the mutation at Ser366
of STING completely abolished IRF3 activation by ISD.
Although we have not detected STING phosphorylation at Ser366 and cannot rule out a role for the phosphorylation of this residue, STING mutants bearing the S366A or L374A mutation are still phosphorylated in response to ISD stimulation. The phosphorylation of STING and TBK1 in cells expressing the S366A or L374A mutant suggest that TBK1 is activated in these cells. Therefore, S366A and L374A mutations selectively inhibit IRF3 phosphorylation by activated TBK1.
Our results not only provide a mechanism of specific phosphorylation of IRF3 in the cytosolic DNA sensing pathway, but also suggest that it is possible to identify inhibitors that selectively inhibit IRF3 without affecting the phosphorylation of other TBK1 substrates. Such inhibitors may prove useful for treating certain human diseases. For example, mice lacking IRF3 or interferon receptors are resistant to the lethal effects of infection by the bacterium Listeria monocytogenes
or by the parasite Plasmodium falciparum
, which causes malaria, the world’s most common infectious disease (12