Here we provide data showing that RIG-I serine 8 is phosphorylated in human cell lines and that this phosphorylation is related to a repressive mechanism that affects the ability of RIG-I to induce IFN-β synthesis. Although phosphorylation is commonly associated with activation mechanisms, there are many examples where phosphorylation of a protein results in its functional repression. Phosphorylation events in the microtubule plus-end tracking proteins inhibit its interaction with EB1, thereby affecting the targeting of plus-end tracking proteins to growing microtubules (33
). NF-κB regulation can also be negatively regulated by phosphorylation of NEMO. Specifically, phosphorylation of serine 68 in the IκB kinase-binding domain of NEMO interferes with the structure of the IκB kinase complex and the tumor necrosis factor-α-induced NF-κB activity (34
By combining mass spectrometry analysis with biochemical and cellular biology experiments, we provide evidence of a new mechanism that may control the IFN-β induction pathway. We show that a negative charge at serine 8 in RIG-I has a negative impact on the ability of TRIM25 to bind the RIG-I CARD functional domain and that it hinders RIG-I CARD ubiquitination and thereby its ability to form a complex with MAVS (). Structural modeling analysis (C
) suggests that serine 8 is an exposed residue and that this residue is in close proximity to the 5-amino acid linker between the two RIG-I CARD-like domains. Because TRIM25 binds to the CARD1 region of RIG-I, we propose that serine 8 phosphorylation regulates the activation of RIG-I by two possible but not mutually exclusive mechanisms. First, serine 8 phosphorylation may affect the ability of the TRIM25 SPRY to bind RIG-I CARD1 and thereby decreases RIG-I CARD ubiquitination. This is consistent with the decrease in the ability of serine 8 phosphomimetic mutants to bind to TRIM25. Second, the close proximity of the phosphorylation site to the C-terminal portion of the CARD1 domain puts this residue in close proximity to the CARD2 of RIG-I, whose lysines 154, 164, and 172 are predicted by structural modeling (data not shown) to be in close proximity to the N terminus of CARD2. These lysines are critical ubiquitination sites for TRIM25 and REUL (20
), and their ubiquitination is required for an optimal RIG-I-mediated IFN-β mRNA induction. The closeness of CARD1 serine 8 to Lys154
, and Lys172
in CARD2 could have a negative impact on the catalytic ubiquitination reaction of these lysines. Thus, unphosphorylated serine 8 in CARD1 may allow both optimal TRIM25 binding and TRIM25-mediated ubiquitination.
A portion of endogenous RIG-I purified from infected and IFN-treated cells was phosphorylated at serine 8, but we found that IFN induction and viral infection lead to lower levels of phosphorylated serine 8 (). Thus, although RIG-I serine 8 phosphorylation may help to maintain an inactive state under non-inducing conditions, stimulation with IFN results not only in increased levels of RIG-I but also in decreased serine 8 phosphorylation, which is consistent with the known positive feedback mechanism of IFN in priming cells to induce higher amounts of IFN when stimulated by virus infection (35
). The S8A RIG-I mutant showed a modest but consistently higher capacity to induce the IFN-β promoter as compared with wild type RIG-I. This increased potential could be an indication of the contribution of the serine 8 phosphorylation to the partial repression of RIG-I. The presence of a serine at position 8 is exclusive to primates, with the other species containing an asparagine at this position (C
). Thus, RIG-I serine 8 phosphorylation appears to be a primate-specific negative regulatory mechanism for the control of RIG-I activity. It remains to be determined whether a different residue is phosphorylated in RIG-I from non-primate species and if that contributes to the negative regulation of IFN in these species.
Overexpressed GST-RIG-I 2CARD contained phosphorylated serine 8 in both ubiquitinated and non-ubiquitinated species, although the ratio between phosphorylated and non-phosphorylated peptides at serine 8 was higher in the non-ubiquitinated CARDs (data not shown). This indicates that RIG-I serine 8 phosphorylation could occur independently of its ubiquitination status, but more experiments will be needed to evaluate whether this is the case.
We do not know at present which mechanism triggers RIG-I serine 8 phosphorylation or dephosphorylation and which cellular kinases and phosphatases are responsible for regulating this phosphorylation. RIG-I serine 8 phosphorylation may also be regulated differentially in different cell types or by yet unknown stimuli, such as cellular stress, hormone environment, or cytokine and chemokine stimulation. We have also found that threonine 170 in the CARD2 domain of RIG-I undergoes phosphorylation and, similar to serine 8 phosphorylation, it has a negative impact on RIG-I ubiquitination and activation (36
). It will then be interesting to know whether RIG-I serine 8 and threonine 170 phosphorylations are controlled by the same or a different set of cellular kinases/phosphatases. Our observations of the impact of phosphorylation on RIG-I activity open a new aspect of RIG-I regulation that will require further research in this area.
In addition to RIG-I phosphorylation described here, ubiquitination and ISGylation have been shown to be involved in the regulation of RIG-I activity (20
polyubiquitination of the RIG-I CARD region has been shown to be essential for the activation of RIG-I. TRIM25 and REUL E3 are the ubiquitin ligases that mediate this ubiquitination, but the mechanism by which these ligases are activated or repressed remains unknown. It has been suggested that the key amino acids in RIG-I CARD for its ubiquitination are exposed following the recognition of viral RNA by RIG-I (7
), but the sequence of these associated events and their molecular regulation are unclear. It is only now becoming clearer that although RIG-I is able to bind several types of RNA, only 5′-triphosphate moieties of RNA (39
) that contain double-stranded regions (4
) are able to trigger RIG-I-mediated induction of IFN-β. In addition, although RIG-I activation is linked to cellular processes triggered by virus infection (40
), the actual activator template of RIG-I in virus-infected cells remains unknown, and it remains unknown whether this activator has to be accompanied by other co-stimuli that may deactivate the RIG-I suppressive mechanisms. In this context, protein phosphatases are likely to play an important role in controlling RIG-I serine 8 phosphorylation. Together, the data we present here on RIG-I repression by serine 8 phosphorylation open a new aspect of the regulation in the IFN pathway that may play an important role in pathological processes related to viral infection and/or autoimmunity.