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Tyrosine phosphorylation of the E3 ubiquitin ligase TRIM21 positively regulates interaction with IRF3 and hence TRIM21 activity.

Stacey KB, Breen E, Jefferies CA - PLoS ONE (2012)

Bottom Line: Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter.Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4.Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department Molecular and Cellular Therapeutics, Royal College Surgeons in Ireland Research Institute, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT
Patients suffering from Systemic Lupus Erythematous (SLE) have elevated type I interferon (IFN) levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs). However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F). We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN-β promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

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Tyrosine 393 regulates IRF3 interaction and hence the activity of TRIM21.(A), Netphos data predicting potential tyrosine phosphorylation sites in TRIM21. (B–G) TRIF-driven IFN-β reporter gene analysis was measured in HEK293T cells transfected with indicated TRIM21 plasmids. Kruskal-Wallis ANOVA analysis assessed relative fold increase of IFN-β promoter activity. * P<0.05 was considered significant and is compared to TRIF driven IFN-β promoter activity alone. (H) Cell lysates prepared from HEK293T cells overexpressing WT TRIM21 and the indicated mutants were assessed for expression of TRIM21 by western blotting with the anti-V5 antibody (Invitrogen). (I) Cell lysates prepared from HEK293T cells overexpressing FLAG-IRF3 were incubated with recombinant GST-PRY/SPRY TRIM21 pre-coupled to GSH-sepharose beads as indicated and the interaction between TRIM21and IRF3 assessed by immunoblotting. Figures are representative of three separate experiments.
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pone-0034041-g003: Tyrosine 393 regulates IRF3 interaction and hence the activity of TRIM21.(A), Netphos data predicting potential tyrosine phosphorylation sites in TRIM21. (B–G) TRIF-driven IFN-β reporter gene analysis was measured in HEK293T cells transfected with indicated TRIM21 plasmids. Kruskal-Wallis ANOVA analysis assessed relative fold increase of IFN-β promoter activity. * P<0.05 was considered significant and is compared to TRIF driven IFN-β promoter activity alone. (H) Cell lysates prepared from HEK293T cells overexpressing WT TRIM21 and the indicated mutants were assessed for expression of TRIM21 by western blotting with the anti-V5 antibody (Invitrogen). (I) Cell lysates prepared from HEK293T cells overexpressing FLAG-IRF3 were incubated with recombinant GST-PRY/SPRY TRIM21 pre-coupled to GSH-sepharose beads as indicated and the interaction between TRIM21and IRF3 assessed by immunoblotting. Figures are representative of three separate experiments.

Mentions: Y393 is important for TRIM21 function– Using NetPhos (http://www.cbs.dtu.dk/services/NetPhos/), tyrosine 343, 388 and 393 were strongly predicted to be phosphorylated (as shown by the NetPhos score, figure 3a) and were subsequently mutated to phenylalanine in order to assess their role in regulating TRIM21 function. Y343 and Y388 are conserved between human and murine TRIM21 and are also predicted to be phosphorylated. Interestingly, Y393 in human TRIM21 corresponds to S396 in murine TRIM21 and this serine residue is also predicted to be phosphorylated in murine TRIM21 (data not shown). As TRIM21 has previously been demonstrated to be a potent inhibitor of TLR-driven IFN-β production [6], we employed an IFN-β promoter reporter gene assay driven by an upstream activator of the pathway, the adaptor protein TRIF, and assessed the effects of each of the tyrosine mutants in this system. As expected from previous reports, wild type full length TRIM21 dose dependently inhibited IFN-β promoter activity, in keeping with its role as a negative regulator of this response (figure 3b). Similarly transfection of cells with increasing amounts of Y343F resulted in a similar inhibitory response, indicating that Y343 is not essential for TRIM21 activity (figure 3c). In contrast we observed a lack of inhibitory activity when Y388F and to a greater extent Y393F mutants were assessed (figure 3d and 3e, respectively). Further analysis to assess the effects of double (Y343,388F) and triple (Y343,388,393F) mutants of TRIM21 was also conducted, with both the double and triple mutants inhibiting the response in a similar manner observed in the Y393F mutant as seen in figure 3f and 3g respectively. To ensure all plasmids were expressing to a similar level, expression analysis of the mutants was carried out. Figure 3h illustrates that Y393F expresses to a higher level than the other TRIM21 plasmids. However the increased expression of Y393F does not lead to an increase in inhibition of IFN-β promoter activity, emphasising that the lack of inhibition in this regard is as a result of the tyrosine to phenylalanine mutation. All other TRIM21 plasmids express to a similar level within the cell, again suggesting that changes observed with these mutants in their ability to inhibit TRIF-driven IFN-β promoter activity is indeed as a result of the tyrosine to phenylalanine mutations and not due to differences in protein expression. These results suggest that tyrosine phosphorylation of Y393 is required for the activity of TRIM21 in this context to be maintained. Previous reports of tyrosine phosphorylation of E3 ligases such as c-Cbl have shown that this modification can either alter intramolecular interactions within the protein or enhance interaction with its substrate [17], [18]. Tyrosine phosphorylation may also alter subcellular localisation of a protein or its stability [29]. Expression analysis of our mutants indicated that stability of TRIM21 was not affected (figure 3h), and given the fact that tyrosine phosphorylation regulated the activity of the E3 ligase and occurred in the PRY/SPRY domain, we hypothesised that phosphorylation at Y393 and possibly Y388 may alter substrate interaction.


Tyrosine phosphorylation of the E3 ubiquitin ligase TRIM21 positively regulates interaction with IRF3 and hence TRIM21 activity.

Stacey KB, Breen E, Jefferies CA - PLoS ONE (2012)

Tyrosine 393 regulates IRF3 interaction and hence the activity of TRIM21.(A), Netphos data predicting potential tyrosine phosphorylation sites in TRIM21. (B–G) TRIF-driven IFN-β reporter gene analysis was measured in HEK293T cells transfected with indicated TRIM21 plasmids. Kruskal-Wallis ANOVA analysis assessed relative fold increase of IFN-β promoter activity. * P<0.05 was considered significant and is compared to TRIF driven IFN-β promoter activity alone. (H) Cell lysates prepared from HEK293T cells overexpressing WT TRIM21 and the indicated mutants were assessed for expression of TRIM21 by western blotting with the anti-V5 antibody (Invitrogen). (I) Cell lysates prepared from HEK293T cells overexpressing FLAG-IRF3 were incubated with recombinant GST-PRY/SPRY TRIM21 pre-coupled to GSH-sepharose beads as indicated and the interaction between TRIM21and IRF3 assessed by immunoblotting. Figures are representative of three separate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316593&req=5

pone-0034041-g003: Tyrosine 393 regulates IRF3 interaction and hence the activity of TRIM21.(A), Netphos data predicting potential tyrosine phosphorylation sites in TRIM21. (B–G) TRIF-driven IFN-β reporter gene analysis was measured in HEK293T cells transfected with indicated TRIM21 plasmids. Kruskal-Wallis ANOVA analysis assessed relative fold increase of IFN-β promoter activity. * P<0.05 was considered significant and is compared to TRIF driven IFN-β promoter activity alone. (H) Cell lysates prepared from HEK293T cells overexpressing WT TRIM21 and the indicated mutants were assessed for expression of TRIM21 by western blotting with the anti-V5 antibody (Invitrogen). (I) Cell lysates prepared from HEK293T cells overexpressing FLAG-IRF3 were incubated with recombinant GST-PRY/SPRY TRIM21 pre-coupled to GSH-sepharose beads as indicated and the interaction between TRIM21and IRF3 assessed by immunoblotting. Figures are representative of three separate experiments.
Mentions: Y393 is important for TRIM21 function– Using NetPhos (http://www.cbs.dtu.dk/services/NetPhos/), tyrosine 343, 388 and 393 were strongly predicted to be phosphorylated (as shown by the NetPhos score, figure 3a) and were subsequently mutated to phenylalanine in order to assess their role in regulating TRIM21 function. Y343 and Y388 are conserved between human and murine TRIM21 and are also predicted to be phosphorylated. Interestingly, Y393 in human TRIM21 corresponds to S396 in murine TRIM21 and this serine residue is also predicted to be phosphorylated in murine TRIM21 (data not shown). As TRIM21 has previously been demonstrated to be a potent inhibitor of TLR-driven IFN-β production [6], we employed an IFN-β promoter reporter gene assay driven by an upstream activator of the pathway, the adaptor protein TRIF, and assessed the effects of each of the tyrosine mutants in this system. As expected from previous reports, wild type full length TRIM21 dose dependently inhibited IFN-β promoter activity, in keeping with its role as a negative regulator of this response (figure 3b). Similarly transfection of cells with increasing amounts of Y343F resulted in a similar inhibitory response, indicating that Y343 is not essential for TRIM21 activity (figure 3c). In contrast we observed a lack of inhibitory activity when Y388F and to a greater extent Y393F mutants were assessed (figure 3d and 3e, respectively). Further analysis to assess the effects of double (Y343,388F) and triple (Y343,388,393F) mutants of TRIM21 was also conducted, with both the double and triple mutants inhibiting the response in a similar manner observed in the Y393F mutant as seen in figure 3f and 3g respectively. To ensure all plasmids were expressing to a similar level, expression analysis of the mutants was carried out. Figure 3h illustrates that Y393F expresses to a higher level than the other TRIM21 plasmids. However the increased expression of Y393F does not lead to an increase in inhibition of IFN-β promoter activity, emphasising that the lack of inhibition in this regard is as a result of the tyrosine to phenylalanine mutation. All other TRIM21 plasmids express to a similar level within the cell, again suggesting that changes observed with these mutants in their ability to inhibit TRIF-driven IFN-β promoter activity is indeed as a result of the tyrosine to phenylalanine mutations and not due to differences in protein expression. These results suggest that tyrosine phosphorylation of Y393 is required for the activity of TRIM21 in this context to be maintained. Previous reports of tyrosine phosphorylation of E3 ligases such as c-Cbl have shown that this modification can either alter intramolecular interactions within the protein or enhance interaction with its substrate [17], [18]. Tyrosine phosphorylation may also alter subcellular localisation of a protein or its stability [29]. Expression analysis of our mutants indicated that stability of TRIM21 was not affected (figure 3h), and given the fact that tyrosine phosphorylation regulated the activity of the E3 ligase and occurred in the PRY/SPRY domain, we hypothesised that phosphorylation at Y393 and possibly Y388 may alter substrate interaction.

Bottom Line: Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter.Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4.Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department Molecular and Cellular Therapeutics, Royal College Surgeons in Ireland Research Institute, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT
Patients suffering from Systemic Lupus Erythematous (SLE) have elevated type I interferon (IFN) levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs). However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F). We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN-β promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

Show MeSH
Related in: MedlinePlus