Co-author YP ABSTRACT M Syk P RESULTS NF SUMMARY B · An ITAM in Reovirus Regulates Activation of...
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An ITAM in Reovirus Regulates Activation of NF-κB , Induction of Interferon-β, and Viral Spread Rachael Stebbing 1 , Susan Irvin 1* , Efraín Rivera Serrano 1 , Karl Boehme 2 , Mine Ikizler 2 , Jeffrey Yoder 1 , Terence Dermody 2 , and Barbara Sherry 1 1 North Carolina State University, Raleigh, NC 27607, 2 Vanderbilt University School of Medicine, Nashville, TN 37232, * Co-author Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains present in the cytoplasmic tails of many surface receptors and transmembrane adaptor proteins that mediate a variety of key cellular responses including immune cell activation. ITAMs have also been identified in enveloped viruses, where they use the same signaling intermediates as cellular ITAMs to participate in pathogenesis and oncogenesis. Our lab identified ITAMs in three mammalian reovirus outer capsid and core proteins, σ2, µ2, and λ2. These are the first ITAMs identified in a nonenveloped virus. ITAM-mediated cell signaling requires the phosphorylation of two tyrosine residues within the motif. To determine the role of ITAMs in reovirus replication and pathogenesis, we used reverse genetics to engineer mutant reoviruses in which two critical tyrosine residues in each ITAM were replaced with phenylalanine to prevent ITAM phosphorylation. While the λ2 mutant virus was not viable, the σ2 and µ2 ITAMs were not required for viral replication in L929 cells or primary cardiac myocyte cultures. Importantly, the μ2 ITAM was found to regulate the activation of NF-κB and influence the induction of interferon-β (IFN-β) in both L929 cells and primary cardiac myocyte cultures; however, the consequences of these μ2 ITAM effects are cell type-specific. In L929 cells, the μ2 ITAM enhances viral fitness. In primary cardiac myocyte cultures where the IFN-β response is critical for antiviral protection and NF-κB is not required for apoptosis, the μ2 ITAM diminishes viral fitness. These results suggest the μ2 ITAM has a cell type-specific role in viral spread, likely reflecting the cell type specific function of NF-κB and IFN-β. We are continuing our studies to provide direct evidence of μ2 ITAM function by investigating phosphorylation states and downstream signaling intermediates of wild type and mutant ITAM viruses. ABSTRACT INTRODUCTION Figure 2. Reovirus proteins contain ITAMs. A) ITAMs of prototype reovirus strains T3D, T2J, and T1L in the σ2, µ2, and λ2 proteins. B) Clustal alignment of the μ2 ITAM with cellular ITAMs. Black indicates conserved ITAM residues. Grey indicates acidic residues common to many ITAMs. A. B. Figure 1. Cellular ITAM activation and downstream signaling. Upon receptor-ligand binding, the ITAM containing protein recruits Src family kinases for phosphorylation of ITAM tyrosine residues. Phosphorylated ITAMs recruit Syk family kinases through binding of tandem SH2 domains of Syk to the two phosphorylated tyrosine residues in the receptor complex. Following ITAM association, Syk autophosphorylates to stimulates downstream signaling. Immunoreceptor tyrosine-based activation motifs (ITAMs) consist of YxxI/Lx 6- 12 YxxI/L sequences. They are most often found in transmembrane and adaptor proteins such as B-cell receptors and DAP12, but they have also been identified in enveloped viruses where they contribute to pathogenesis and oncogenesis. ITAM- mediated cell signaling requires the phosphorylation of two tyrosine residues within the motif (Figure 1). In this study we identified ITAMs in the reovirus σ2, µ2, and λ2 proteins encoded by the S2, M1, and L2 gene segments (Figure 2). To determine whether ITAMs function in reovirus replication, plasmid-based reverse genetics was employed to engineer WT reovirus strain T3D mutant viruses (YYFF series) in which the two critical ITAM tyrosine residues (YY) were exchanged with phenylalanine (FF) to prevent phosphorylation required for ITAM function (Figure 2). Src Family Kinases NFκB NFAT MAPKs Syk P P P Cellular Cytokine Proliferation Cell Differentiation Release Death Y Y ITAM SUMMARY Figure 9. Effect of μ2 ITAM on reovirus replication and cytopathic effect is cell-type specific. In L929 cells, μ2 ITAM activation of NF-κB participates in reovirus-induced apoptosis, enhancing viral spread. In cardiac myocytes, where the IFN response is critical to limit viral damage and where NF-κB is not required for apoptosis, the μ2 ITAM diminishes viral replication and spread. Figure 10. Possible mechanisms for μ2 ITAM activation of Syk. The μ2 protein does not span membranes, suggesting it functions in a manner that differs from other ITAM- containing proteins. One possible mechanism is that Syk is recruited to viral factories where μ2 is abundant and highly concentrated in a membrane bound inclusion body. • The μ2 ITAM regulates the induction of Interferon-β. Despite replicating to equivalent or higher titers, μ2-YYFF induced significantly less IFN-β than WT T3D in L929 cells and primary cardiac myocyte cultures. • The μ2 ITAM regulates activation of NF-κB. Reovirus activates NF-κB during infection, inducing apoptosis in the brain and an IFN-β response in the heart. Both viral infection and ectopically expressed μ2 protein activated NF-κB. In contrast, μ2- YYFF activation of NF-κB was minimal, indicating that the μ2 ITAM is required for μ2 maximal activation of NF-κB. • The μ2 ITAM has a cell-type specific role in viral spread, likely reflecting the cell- type specific functions of NF-κB and IFN-β (Figure 9). • μ2 is phosphorylated on multiple tyrosine residues. Both μ2 and μ2-YYFF are phosphorylated on tyrosine residues, with no detectable difference between the two by Western Blot despite μ2-YYFF missing the two ITAM tyrosines. This is not surprising given that there are 30 tyrosine residues in μ2, and any of them may be phosphorylated at varying frequencies and for varying lengths of time. • The μ2 ITAM activates NF-κB through the ITAM signaling intermediate Syk. In the presence of the Syk specific inhibitor BAY 61-3606, NF-κB activation by μ2 was significantly reduced, while μ2-YYFF activation of NF-κB remained minimal in the presence or absence of the inhibitor. • The μ2 ITAM activates the ITAM signaling intermediate, Syk. Cellular ITAMs recruit and activate Syk to signal to downstream molecules including NF-κB. μ2 induced Syk phosphorylation while the mutant, μ2-YYFF, did not, providing direct evidence that μ2 has a functional ITAM. Possible mechanisms for μ2 activation of Syk are outlined in Figure 10. Membrane-associated ITAMs Reovirus μ2 ITAM NFκB L929 Cells Cardiac Myocytes Apoptosis Increased CPE and Viral Spread Decreased CPE and Viral Spread IFNβ ACKNOWLEDGEMENTS We thank Ralph Baric, Lianna Li, Ray Pickles, Frank Scholle, Jennifer Zurney, Kim Parks, and Lance Johnson for many helpful discussions and outstanding technical assistance. This research was supported by Public Health Service awards T32 CA09385 (K.W.B.), F32 AI075776 (K.W.B), R37 AI38296 (T.S.D), R01 AI50080 (T.S.D), and R01 A1083333 (B.S) and the Elizabeth B. Lamb Center for Pediatric Research. Src Family Kinases NFκB Syk P P P Y Y ITAM μ2 μ2 Syk P μ2 NFκB μ2 RESULTS Figure 3. The μ2 ITAM regulates induction of IFNβ. WT T3D, σ2-YYFF, or μ2-YYFF viruses were added to L929 cells or cardiac myocytes. 8 or 10 h post-infection, RNA was quantified by reverse transcription and quantitative RealTime PCR , and copy number was normalized to GAPDH. In either cell type, μ2-YYFF induced significantly less IFNβ than WT or σ2-YYFF despite replication to equivalent or higher titers. Figure 4. The μ2 ITAM effect on cytopathic effect is cell type specific. L929 cells or cardiac myocytes were infected with low multiplicities of WT T3D, σ2-YYFF, or μ2-YYFF viruses and after multiple cycles of infection and spread, cell viability was quantified by MTT assay. In L929 cells, μ2-YYFF was significantly less cytopathic (leaving more cells viable) than WT or σ2-YYFF. In contrast, μ2-YYFF was significantly more cytopathic than WT or σ2-YYFF in primary cardiac myocyte cultures. Figure 7. μ2 is phosphorylated on multiple tyrosine residues. AD-293 cells were transfected with the indicated FLAG-tagged plasmid (HCV NS3/4A is a negative control that is not phosphorylated). 40 h post-transfection, whole-cell lysates were immunoprecipitated using anti-FLAG beads and fractions were resolved by SDS-PAGE and immunoblotted using mouse anti-FLAG or anti-phosphorylated tyrosine antibodies. This provides the first evidence that μ2 is a phosphorylated protein. However, phosphorylation of μ2- YYFF in the context of 30 μ2 tyrosines prevented concluding whether ITAM tyrosines are phosphorylated. Figure 8. μ2 activates Syk, proving that the μ2 ITAM functions as an ITAM. AD-293 cells were co-transfected with Syk and the indicated FLAG-tagged plasmid. 40 h post- transfection, whole-cell lysates were resolved by SDS-PAGE and immunoblotted using mouse anti-Syk (A) or rabbit anti-phosphorylated Syk (B) antibodies. As expected, the constitutively active form of the upstream adaptor protein DAP12 induced phosphorylation of the ITAM signaling intermediate Syk while the negative control STAT2 did not. The reovirus protein μ2 also activated Syk, while μ2 –YYFF (lacking the critical ITAM tyrosine residues) did not, demonstrating that the μ2 ITAM functions as an ITAM. A) B) pSyk μ2 μ2-YYFF STAT2 actDAP12 WB: α-pSyk WB: α-Syk Total Syk STAT2 μ2 μ2-YYFF actDAP12 FLAG-NS3/4A FLAG-μ2 IP: anti-FLAG WB: anti-FLAG HCV NS3/4A μ2 μ2-YYFF IP: anti-FLAG WB: anti-pTyr FLAG-NS3/4A FLAG-μ2 HCV NS3/4A μ2 μ2-YYFF Syk Reovirus μ2 merge FLAG- vector FLAG-μ2 FLAG- μ2-YYFF Figure 6. Syk is recruited to viral factories. Vero cells were co-transfected with the indicated FLAG-tagged plasmid, reovirus protein μNS to induce viral factory formation, and Syk. 24 h post transfection, cells were fixed and immunolabeled with an antibody against Syk (green) and an antibody against reovirus protein μ2 (red). (Zeiss LMS 710 confocal microscope, 40x). Syk is known to be recruited to ITAMs following phosphorylation of the two critical ITAM tyrosine residues. In cells transfected with μ2, Syk was localized near viral factories while in cells transfected with μ2-YYFF, Syk remained diffuse throughout the cell. Results suggest Syk interacts with the μ2 ITAM in viral factories where μ2 is concentrated within the cell. FLAG-μ2 FLAG-μ2-YYFF FLAG-μ2 FLAG-μ2-YYFF 1 μg DNA/well 0.1 μg DNA/well Figure 5. The μ2 ITAM regulates activation of NF-κB. A) HEK-293 cells were transfected with constitutively-expressing Renilla-luciferase plasmid as an internal standard, and an NF-κB-luciferase reporter plasmid and then infected with the indicated virus. Luciferase activity was quantified. The μ2- YYFF virus activated significantly less NF- κB than did either WT or σ2-YYFF virus. B) Cells were transfected as for (A) but transfections included the indicated FLAG-tagged plasmid. At 24 hours post-transfection, luciferase activity was quantified. Over-expressed FLAG-μ2 protein alone activated NF-κB, indicating that μ2 can activate NF-κB in the absence of viral infection. FLAG-μ2-YYFF protein activation of NF-κB was minimal or undetectable, indicating that the μ2 ITAM is required for μ2 maximal activation of NF-κB. As a control, Corresponding whole-cell lysates were resolved by SDS-PAGE and immunoblotted using mouse anti-FLAG or anti-GAPDH antibodies. Even when levels of FLAG-μ2-YYFF were greater than five-fold higher than those of FLAG-μ2, FLAG-μ2-YYFF activation of NF-κB was significantly less than that for FLAG-μ2. C) AD-293 cells were pretreated with DMSO alone or 10 μM of the Syk-specific inhibitor BAY 61-3606 and then cotransfected with the indicated FLAG-tagged plasmid and Syk, a downstream signaling intermediate in the ITAM pathway. At 24 hours post-transfection, luciferase activity was quantified. NF-κB activation by μ2 was significantly reduced by BAY 61-3606 , μ2-YYFF NF-κB activation remained minimal in the presence or absence of the inhibitor, and TNFα activation of NF-κB was unaffected by the inhibitor, indicating the μ2 ITAM activates NF-κB through the ITAM signaling intermediate Syk. B. A. Flag C. GAPDH
Transcript of Co-author YP ABSTRACT M Syk P RESULTS NF SUMMARY B · An ITAM in Reovirus Regulates Activation of...
An ITAM in Reovirus Regulates Activation of NF-κB, Induction of Interferon-β, and Viral Spread Rachael Stebbing1, Susan Irvin1*, Efraín Rivera Serrano1, Karl Boehme2, Mine Ikizler2, Jeffrey Yoder1, Terence Dermody2, and Barbara Sherry1
1North Carolina State University, Raleigh, NC 27607, 2Vanderbilt University School of Medicine, Nashville, TN 37232, *Co-author
Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains present in the cytoplasmic tails of many surface receptors and transmembrane adaptor proteins that mediate a variety of key cellular responses including immune cell activation. ITAMs have also been identified in enveloped viruses, where they use the same signaling intermediates as cellular ITAMs to participate in pathogenesis and oncogenesis. Our lab identified ITAMs in three mammalian reovirus outer capsid and core proteins, σ2, µ2, and λ2. These are the first ITAMs identified in a nonenveloped virus. ITAM-mediated cell signaling requires the phosphorylation of two tyrosine residues within the motif. To determine the role of ITAMs in reovirus replication and pathogenesis, we used reverse genetics to engineer mutant reoviruses in which two critical tyrosine residues in each ITAM were replaced with phenylalanine to prevent ITAM phosphorylation. While the λ2 mutant virus was not viable, the σ2 and µ2 ITAMs were not required for viral replication in L929 cells or primary cardiac myocyte cultures. Importantly, the µ2 ITAM was found to regulate the activation of NF-κB and influence the induction of interferon-β (IFN-β) in both L929 cells and primary cardiac myocyte cultures; however, the consequences of these µ2 ITAM effects are cell type-specific. In L929 cells, the µ2 ITAM enhances viral fitness. In primary cardiac myocyte cultures where the IFN-β response is critical for antiviral protection and NF-κB is not required for apoptosis, the µ2 ITAM diminishes viral fitness. These results suggest the µ2 ITAM has a cell type-specific role in viral spread, likely reflecting the cell type specific function of NF-κB and IFN-β. We are continuing our studies to provide direct evidence of µ2 ITAM function by investigating phosphorylation states and downstream signaling intermediates of wild type and mutant ITAM viruses.
ABSTRAC T
INTRODUC TION
Figure 2. Reovirus proteins contain ITAMs. A) ITAMs of prototype reovirus strains T3D, T2J, and T1L in the σ2, µ2, and λ2 proteins. B) Clustal alignment of the µ2 ITAM with cellular ITAMs. Black indicates conserved ITAM residues. Grey indicates acidic residues common to many ITAMs.
A. B.
Figure 1. Cellular ITAM activation and downstream signaling. Upon receptor-ligand binding, the ITAM containing protein recruits Src family kinases for phosphorylation of ITAM tyrosine residues. Phosphorylated ITAMs recruit Syk family kinases through binding of tandem SH2 domains of Syk to the two phosphorylated tyrosine residues in the receptor complex. Following ITAM association, Syk autophosphorylates to stimulates downstream signaling.
Immunoreceptor tyrosine-based activation motifs (ITAMs) consist of YxxI/Lx6-
12YxxI/L sequences. They are most often found in transmembrane and adaptor proteins such as B-cell receptors and DAP12, but they have also been identified in enveloped viruses where they contribute to pathogenesis and oncogenesis. ITAM-mediated cell signaling requires the phosphorylation of two tyrosine residues within the motif (Figure 1). In this study we identified ITAMs in the reovirus σ2, µ2, and λ2 proteins encoded by the S2, M1, and L2 gene segments (Figure 2). To determine whether ITAMs function in reovirus replication, plasmid-based reverse genetics was employed to engineer WT reovirus strain T3D mutant viruses (YYFF series) in which the two critical ITAM tyrosine residues (YY) were exchanged with phenylalanine (FF) to prevent phosphorylation required for ITAM function (Figure 2).
SrcFamily Kinases
NFκB
NFATMAPKs
Syk PP
P
Cellular Cytokine Proliferation CellDifferentiation Release Death
Y
YITA
M
SUMMARY
Figure 9. Effect of µ2 ITAM on reovirus replication and cytopathic effect is cell-type specific. In L929 cells, µ2 ITAM activation of NF-κB participates in reovirus-induced apoptosis, enhancing viral spread. In cardiac myocytes, where the IFN response is critical to limit viral damage and where NF-κB is not required for apoptosis, the µ2 ITAM diminishes viral replication and spread.
Figure 10. Possible mechanisms for µ2 ITAM activation of Syk. The µ2 protein does not span membranes, suggesting it functions in a manner that differs from other ITAM- containing proteins. One possible mechanism is that Syk is recruited to viral factories where µ2 is abundant and highly concentrated in a membrane bound inclusion body.
• The µ2 ITAM regulates the induction of Interferon-β. Despite replicating to equivalent or higher titers, µ2-YYFF induced significantly less IFN-β than WT T3D in L929 cells and primary cardiac myocyte cultures. • The µ2 ITAM regulates activation of NF-κB. Reovirus activates NF-κB during infection, inducing apoptosis in the brain and an IFN-β response in the heart. Both viral infection and ectopically expressed µ2 protein activated NF-κB. In contrast, µ2-YYFF activation of NF-κB was minimal, indicating that the µ2 ITAM is required for µ2 maximal activation of NF-κB. • The µ2 ITAM has a cell-type specific role in viral spread, likely reflecting the cell-type specific functions of NF-κB and IFN-β (Figure 9).
• µ2 is phosphorylated on multiple tyrosine residues. Both µ2 and µ2-YYFF are phosphorylated on tyrosine residues, with no detectable difference between the two by Western Blot despite µ2-YYFF missing the two ITAM tyrosines. This is not surprising given that there are 30 tyrosine residues in µ2, and any of them may be phosphorylated at varying frequencies and for varying lengths of time. • The µ2 ITAM activates NF-κB through the ITAM signaling intermediate Syk. In the presence of the Syk specific inhibitor BAY 61-3606, NF-κB activation by µ2 was significantly reduced, while µ2-YYFF activation of NF-κB remained minimal in the presence or absence of the inhibitor. • The µ2 ITAM activates the ITAM signaling intermediate, Syk. Cellular ITAMs recruit and activate Syk to signal to downstream molecules including NF-κB. µ2 induced Syk phosphorylation while the mutant, µ2-YYFF, did not, providing direct evidence that µ2 has a functional ITAM. Possible mechanisms for µ2 activation of Syk are outlined in Figure 10.
Membrane-associated ITAMs Reovirus µ2 ITAM
NFκB
L929 Cells Cardiac Myocytes
Apoptosis
Increased CPE and Viral Spread
Decreased CPE and Viral Spread
IFNβ
ACKNOWLEDGEMENTS We thank Ralph Baric, Lianna Li, Ray Pickles, Frank Scholle, Jennifer Zurney, Kim Parks, and Lance Johnson for many helpful discussions and outstanding technical assistance. This research was supported by Public Health Service awards T32 CA09385 (K.W.B.), F32 AI075776 (K.W.B), R37 AI38296 (T.S.D), R01 AI50080 (T.S.D), and R01 A1083333 (B.S) and the Elizabeth B. Lamb Center for Pediatric Research.
SrcFamily Kinases
NFκB
Syk P
P
P
Y
YITA
M
µ2
µ2
Syk Pµ2
NFκB
µ2
RESULTS
Figure 3. The µ2 ITAM regulates induction of IFNβ. WT T3D, σ2-YYFF, or µ2-YYFF viruses were added to L929 cells or cardiac myocytes. 8 or 10 h post-infection, RNA was quantified by reverse transcription and quantitative RealTime PCR , and copy number was normalized to GAPDH. In either cell type, µ2-YYFF induced significantly less IFNβ than WT or σ2-YYFF despite replication to equivalent or higher titers.
Figure 4. The µ2 ITAM effect on cytopathic effect is cell type specific. L929 cells or cardiac myocytes were infected with low multiplicities of WT T3D, σ2-YYFF, or µ2-YYFF viruses and after multiple cycles of infection and spread, cell viability was quantified by MTT assay. In L929 cells, µ2-YYFF was significantly less cytopathic (leaving more cells viable) than WT or σ2-YYFF. In contrast, µ2-YYFF was significantly more cytopathic than WT or σ2-YYFF in primary cardiac myocyte cultures.
Figure 7. µ2 is phosphorylated on multiple tyrosine residues. AD-293 cells were transfected with the indicated FLAG-tagged plasmid (HCV NS3/4A is a negative control that is not phosphorylated). 40 h post-transfection, whole-cell lysates were immunoprecipitated using anti-FLAG beads and fractions were resolved by SDS-PAGE and immunoblotted using mouse anti-FLAG or anti-phosphorylated tyrosine antibodies. This provides the first evidence that µ2 is a phosphorylated protein. However, phosphorylation of µ2-YYFF in the context of 30 µ2 tyrosines prevented concluding whether ITAM tyrosines are phosphorylated.
Figure 8. µ2 activates Syk, proving that the µ2 ITAM functions as an ITAM. AD-293 cells were co-transfected with Syk and the indicated FLAG-tagged plasmid. 40 h post-transfection, whole-cell lysates were resolved by SDS-PAGE and immunoblotted using mouse anti-Syk (A) or rabbit anti-phosphorylated Syk (B) antibodies. As expected, the constitutively active form of the upstream adaptor protein DAP12 induced phosphorylation of the ITAM signaling intermediate Syk while the negative control STAT2 did not. The reovirus protein µ2 also activated Syk, while µ2 –YYFF (lacking the critical ITAM tyrosine residues) did not, demonstrating that the µ2 ITAM functions as an ITAM.
A) B)
pSyk
µ2µ2-YYFFSTAT2actDAP12
WB: α-pSykWB: α-Syk
Total Syk
STAT2 µ2 µ2-YYFF actDAP12
FLAG-NS3/4A
FLAG-µ2 IP: anti-FLAGWB: anti-FLAG
HCV NS3/4A µ2 µ2-YYFF
IP: anti-FLAGWB: anti-pTyrFLAG-NS3/4A
FLAG-µ2
HCV NS3/4A µ2 µ2-YYFF
Syk Reovirus µ2 merge
FLAG- vector
FLAG-µ2
FLAG- µ2-YYFF
Figure 6. Syk is recruited to viral factories. Vero cells were co-transfected with the indicated FLAG-tagged plasmid, reovirus protein µNS to induce viral factory formation, and Syk. 24 h post transfection, cells were fixed and immunolabeled with an antibody against Syk (green) and an antibody against reovirus protein µ2 (red). (Zeiss LMS 710 confocal microscope, 40x). Syk is known to be recruited to ITAMs following phosphorylation of the two critical ITAM tyrosine residues. In cells transfected with µ2, Syk was localized near viral factories while in cells transfected with µ2-YYFF, Syk remained diffuse throughout the cell. Results suggest Syk interacts with the µ2 ITAM in viral factories where µ2 is concentrated within the cell.
FLAG-µ2 FLAG-µ2-YYFF FLAG-µ2 FLAG-µ2-YYFF
1 µg DNA/well 0.1 µg DNA/well
Figure 5. The µ2 ITAM regulates activation of NF-κB. A) HEK-293 cells were transfected with
constitutively-expressing Renilla-luciferase plasmid as an internal standard, and an NF-κB-luciferase reporter plasmid and then infected with the indicated virus. Luciferase activity was quantified. The µ2-YYFF virus activated significantly less NF-κB than did either WT or σ2-YYFF virus.
B) Cells were transfected as for (A) but transfections included the indicated FLAG-tagged plasmid. At 24 hours post-transfection, luciferase activity was quantified. Over-expressed FLAG-µ2 protein alone activated NF-κB, indicating that µ2 can activate NF-κB in the absence of viral infection. FLAG-µ2-YYFF protein activation of NF-κB was minimal or undetectable, indicating that the µ2 ITAM is required for µ2 maximal activation of NF-κB. As a control, Corresponding whole-cell lysates were resolved by SDS-PAGE and immunoblotted using mouse anti-FLAG or anti-GAPDH antibodies. Even when levels of FLAG-µ2-YYFF were greater than five-fold higher than those of FLAG-µ2, FLAG-µ2-YYFF activation of NF-κB was significantly less than that for FLAG-µ2.
C) AD-293 cells were pretreated with DMSO alone or 10 µM of the Syk-specific inhibitor BAY 61-3606 and then cotransfected with the indicated FLAG-tagged plasmid and Syk, a downstream signaling intermediate in the ITAM pathway. At 24 hours post-transfection, luciferase activity was quantified. NF-κB activation by µ2 was significantly reduced by BAY 61-3606 , µ2-YYFF NF-κB activation remained minimal in the presence or absence of the inhibitor, and TNFα activation of NF-κB was unaffected by the inhibitor, indicating the µ2 ITAM activates NF-κB through the ITAM signaling intermediate Syk.
B. A.
Flag
C.
GAPDH
