The Role Of Ubiquitin Ligase Smurf1in Regulating Interferon Signaling

ObjectiveInterferons (IFNs) are important cytokines that play essential roles in antiviral, antibacterial, antitumor and immunomodulatory activities. IFNs primarily signals through the JAK-STAT pathway leading to the activation of signal transducer and activator of transcription (STAT) and subsequent transcription of target genes. Previous studies reported that uncontrolled IFNs signaling may lead to many immune disorders. However, the mechanisms that regulate IFNs signaling are still unclear.As a HECT-type E3ligase, Smad ubiquitination regulation factor1(Smurfl) targets many proteins for ubiquitination and degradation. Smurfl was originally identified as the interacted protein of Smadl in1992, and subsequent studies found that Smurfl plays a critical role in the regulation of TGF-β signaling pathway by targeting the degradation of Smadl/5, TGFβR, RhoA, MEKK2, Prickle1and JunB. Although the functions of Smurfl in the regulation of TGF-β signaling are well defined, other cellular signaling pathways especially in the immune responses regulated by Smurfl are not clear. In this regard, two recent studies have demonstrated that Smurfl can regulate immune response. One study showed that Smurfl can mediate TRAFs, which play important roles in immune response, ubiquitination and degradation. The other study showed that Smurfl, which forms a complex together with Smurf2and Smad6, negatively regulate TLR signaling pathway by promoting MyD88degradation.To investigate the role of Smurfl in immune response, we studied the effects of Smurfl on IFNs signaling, and uncovered the molecular mechanism involved.Methods1. We transfected mouse leukaemic monocyte macrophage cell line RAW264.7with Smurfl overexpression plasmids, then detected the effects of Smurfl on the activity of GAS-luc report gene induced by IFN-y using Dual-Luciferase reporter assay system, and the effects of Smurfl on the activity of iNos gene promoter.2. We transfected mouse peritoneal macrophages with Smurfl-specified siRNA, then detected the effects of Smurfl on the expression of IFNs target gens including iNOS, IRF1, Cxcl9, Cxcl10, through quantitative real time polymerase chain reaction.3. We investigated the interaction between Smurfl and STAT1through co-immunoprecipitation and immunofluorescence. Then we used various Smurfl and STAT1mutants to map the domain or motif required for the interaction between Smurfl and STAT1.4. We investigated the effects of Smurfl on the protein level and ubiquitination of STAT1through cotransfection and in vivo ubiquitination assays. Then we used STAT1phosphorylation site mutants to study whether STAT1ubiquitination mediated by Smurfl is dependent on its phosphorylation.5. We explored the role of Smurfl in anti-viral immune response mediated by IFN-y through VSV infection and plaque assays.6. In order to detect the effects of IFNs on the expression of Smurfl, RAW264.7cells, mouse peritoneal macrophages, NIH3T3cells, and HEK293cells were respectively treated with IFN-β and IFN-y, then we measured the expression of Smurfl through Western blot.Results1. Smurfl negatively regulates IFNs signalingTo explore the functions of Smurfl inimmune response, we initially investigated the effect of Smurfl on interferon signaling. To investigate the role of Smurfl in IFNs signaling, the effect of Smurfl on the activation of the STAT1cis-reporting plasmid GAS-Luc was investigated. Using dual-Luciferase reporter assay system we found that overexpression of Smurfl significantly inhibited IFN-y-induced transcriptional activation of GAS-Luc reporter plasmids. Furthermore, Smurfl inhibited IFN-y-induced activation of GAS-Luc reporter in dose-dependent manner. Similarly, Smurfl also significantly inhibited IFN-y-induced transcriptional activation of iNos gene promoter in a dose-dependent manner.To directly examine the function of Smurfl in IFN-y-mediated gene expression under physiological conditions, mouse peritoneal macrophages were transfected with control siRNA or Smurfl-specific siRNA to silence Smurfl expression, then treated the cells with IFN-y for various times. The expression of IFN-y target genes were measured through qRT-PCR. We found that IFN-γ-induced expression of Cxcl9, Cxcl10, Irfl and Nos2was significantly enhanced in Smurfl siRNA transfected macrophages. Similarly, IFN-β-induced expression of Cxcl9, Cxcl10, Ly6e and Ifi203was also significantly enhanced in Smurfl siRNA-transfected macrophages. AII together, these results suggest that Smurfl negatively regulates IFNs signaling.2. Smurfl physically interacts with STAT1Smurfl can inhibit both IFN-β signaling and IFN-y signaling, and the common adaptor downstream of IFN-β and IFN-y is STAT1. Moreover, STAT1has been reported to interact with Smurfl in a large scale luminescence-based mammalian interactome mapping. Thus, we hypothesized that Smurfl negatively regulates IFNs signaling by targeting STAT1.To confirm the interaction between Smurfl and STAT1, overexpression plasmids of Smurfl and STAT1were cotransfected into HEK293cells, followed with coimmunoprecipitation and western blot with indicated antibodies. The results showed that exogenous Smurfl could interact with exogenous STAT1. Then the interaction between endogenous Smurfl and endogenous STAT1independent of IFN-y treatment was confirmed in mouse macrophages.It has been reported that Smufl interacts with its target proteins through the WW domains of Smurfl and the proline-rich (PPXY) motif in the substrate proteins. To map the Smufl domains required for the interaction with STAT1, various Smurfl mutants were cotransfected into HEK293cells with STAT1, followed with coimmunoprecipitation. Co-IP assays indicated that the WW domains of Smurfl mediated the interaction with STAT1. Sequence analysis showed that there is a putative PY motif (PLKY amino acid from663to666) in STAT1. In order to determine whether the PY motif of STAT1is required for the interaction with Smurfl, the PLKY motif was mutated into ALKA (STAT1PY/AA). In contrast to WT STAT1, STAT1PY/AA lost the ability to interact with Smurfl as measured with co-IP assays, suggesting the PY motif is required for the interaction with Smurfl. Taken together, these data indicate that Smurfl interacts with STAT1through the WW domains of Smurfl and the PY motif of STAT1.3. Smurfl promoted ubiquitination and degradation of STAT1As an E3ligase, Smurfl mediates the degradation of target proteins. The interaction between Smurfl and STAT1prompted us to investigate the function of Smurfl on the degradation of STAT1. Increasing concentration of Smurfl expression plasmid was cotransfected into HEK293cells together with constant concentration of STAT1expression plasmid. Western blot assays showed that STAT1protein expression was substantially decreased with transfection of Smurfl expression plasmid. In sharp contrast, Smurfl CA mutant could not decrease the STAT1protein level. Similarly, Smurfl transfection could decrease the level of endogenous STAT1protein. To determine if Smurfl inhibits STAT1protein expression under physiological conditions, Smurfl siRNA was transfected into mouse primary peritoneal macrophages, and STAT1protein expression was measured. Transfection of Smurfl siRNA significantly increased total STAT1protein level in macrophages, compared to control siRNA transfected cells. These data suggest that Smurfl could affect STAT1protein level, and this effect require the E3ligase activity of Smurfl.Since Smurfl is an E3ubiquitin ligase and decrease STAT1protein level. Therefore, we hypothesized that Smurfl targets STAT1ubiquitination. To certify our hypothesis, STAT1ubiquitination mediated by Smurfl was measured through in vivo ubiquitination assay. We found that STAT1polyubiquitination was greatly increased in the presence of Smurfl, whereas transfection of Smurfl CA mutant could not increase STAT1polyubiquitination. Previous studies reported that the protein can undergo different types of ubiquitination, and the ubiquitination format determines the fate of protein. Then we detected the form of ubiquitin chains linked to STAT1using ubiquitin mutants and in vivo ubiquitination assays.Immunoprecipitation and western blot analysis showed that Smurfl induced K48-linked polyubiquitination of STAT1, but not K63-linked polyubiquitination. K48-linked protein ubiquitination leads to the degradation of the corresponding protein by26S proteasome. Consistently, Smurfl induced degradation of STAT1protein could be reversed by proteasome inhibitor MG132. Taken together, these results indicate that Smurfl promotes K48-linked polyubiquitination and subsequent proteasomal degradation of STAT1.4. STAT1phosphorylation is not required for Smurfl-mediated STAT1degradationThe phosphorylation on tyrosine701and serine727play an important role in activities of STAT1in transcription. Previous study reported that STAT1ubiquitination mediated by SLIM requires its phosphorylation. Next we investigated whether STAT1degradation mediated by Smurfl requires phosphorylation. Expression plasmids for STAT1wild-type or mutants (Y701F or S727A) were transfected into HEK293together with increasing amounts of Smurfl, the level of STAT1proteins was measured with western blot. Smurfl could induce the degradation of STAT1WT, STAT1mutants Y701F and S727A. Consistently, Smurfl could promote polyubiquitination of STAT1WT, STAT1mutants Y701F and S727A. These data indicated that the degradation of STAT1mediated by Smurfl is independent of tyrosine and serine phosphorylation.5. Smurfl negatively regulates anti-viral immune responsesIFNs have an important function in anti-viral immune response, and Smurfl is a physiological suppressor of IFNs signaling. Since Smurfl is a physiological suppressor of IFN-y signaling, we speculate Smurfl may play a role in the cellular anti-viral response. To directly investigate the effect of Smurfl on IFN-y-mediated antiviral responses, vesicular stomatitis virus (VSV) was used to infect HEK293cells which were transfected with Smurfl overexpression plasmids, and mouse peritonealmacrophages in which Smurfl was silenced. Then the VSV replication was measured through plaque assay and qRT-PCR. Plaque assay of HEK293cells infected with VSV showed that overexpression of Smurfl substantially increased viral replication. In sharp contrast, Smurfl mutant CA could not increase viral replication. Similarly, VSV RNA replication in HEK293cells was sinificantly increased in Smurfl-transfected cells, compared to control vector-or Smurfl CA-transfected cells as measured by quantitative RT-PCR. In order to further confirm the function of Smurfl on VSV replication under physiological condition, Smurfl expression was silenced by Smurfl siRNA transfection in mouse peritoneal macrophages, then the macrophages were stimulated with IFN-γ and infected with VSV. Plaque assay showed that transfection of Smurfl siRNA greatly decreased VSV viral replication in macrophages. Accordingly, knockdown of Smurfl significantly decreased intracellular VSV RNA replication, compared to control siRNA-transfected macrophages. Taken together, these data demonstrate that Smurfl negatively regulates cellular anti-viral immune responses.6. IFN-γ induces the expression of SmurflHaving established the negative role of Smurfl in IFNs signaling, we next examined the effect of IFNs on Smurfl expression. RAW264.7macrophages, mouse peritoneal macrophages, NIH3T3cells, and HEK293cells were respectively treated with IFN-β or IFN-γ for various times and the level of Smurfl protein was analyzed with western blot. The expression level of Smurfl protein was significantly increased after IFN-γ stimulation in these cells, whereas IFN-β could not induce Smurfl expression. Conclusion1. The ubiquitin ligase Smurfl negatively regulates IFNs signaling through promoting STAT1ubiquitination and degradation.2. Smurfl interacts with STAT1through the WW domains of Smurfl and the PY motif of STAT1, and induces STAT1K48-linked poly ubiquitination which is recognized by26S proteasome for degradation.3. Smurfl negatively regulates anti-viral immune responses.4. IFN-y induces the expression of Smurfl which acts as a negative feedback regulator for IFN-y signaling.Innovation and Significance1. In our study, we found that the HECT-type E3ubiquitin ligase Smurfl can negatively regulate IFNs signaling for the first time. These results enrich our understanding of the role of Smurfl in immune response.2. In this study, we elucidated a possible molecular mechanism for the suppression effect of Smurfl on IFNs signaling. We established STAT1as a new target, which can be recognized and ubiquitinated by Smurfl. The discovery widens the substrate spectrum of Smurfl, and proves that there is another E3ubiquitin ligase which can target STAT1ubiquitination besides SLIM.3. Previous study reported that STAT1ubiquitination mediated by SLIM requires its phosphorylation. Interestingly, we found that STAT1phosphorylation is not required for Smurfl-mediated STAT1ubiquitination. Thus, Smurfl may be a global regulator for STAT1.4. Our study found that IFN-y induces the expression of Smurfl which negatively regulate IFN-y signaling by promoting STAT1degradation. This is a new negative feedback regulation mechanism for IFN-y signaling. Limitations of This Study1. Although we found that Smurfl can target STAT1ubiquitination, the ubiquitination sites are still not mapped.2. In addition to the anti-viral function, IFNs play an important role in immune regulation. It requires further research whether Smurfl participates in immune regulation through affecting the IFNs signaling.