Phospho-SXXE/D Motif Mediated TNF Receptor 1–TRADD Death Domain Complex Formation for T Cell Activation and Migration

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1–TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase β plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.

T umor necrosis factor is a proinflammatory cytokine, which can target its two cognate receptors and initiate the activation of NF-kB, caspase, and the JNK pathways, leading to immune cell gene regulation, apoptosis, and/or their immune cell activation. TNF-bound TNFR1 recruits TNFR-associated death domain protein (TRADD), an adaptor protein that serves as the platform for additional recruitment of receptor-interacting protein kinase (RIP) and Fas-associated death domain protein (FADD), initiating both NF-kB activation and apoptosis induction (1)(2)(3). The C terminals of TNFR1, TRADD, FADD, and RIP all carry a discrete region termed the "death domain," which is composed of six continuous a-helical bundles and responsible for homotypic interactions among these four proteins. The death domain has ∼80 to ∼100 aa, similar in size to the Src homology 2 (SH2) domain, both of which can be packed into a similar globular structure. Although the phosphotyrosine within the YXXL/I/V/Q/ M sequence can be recognized by the SH2 domain during proteinprotein interaction, it has been shown that the hydrophobic residue at the p+3 position is also important for SH2 domain recognition, as revealed by synthesized peptide binding analysis (4).
For SH2 domain-bearing proteins, such as growth factor receptor adaptor proteins or transcription factors like STATs, interactions between them and their substrates are cytokine or growth factor stimulation dependent. In the same way, for death domain proteins, the formation of the death domain complex is TNF stimulation dependent. One possible mechanism controlling this modular interaction is death domain phosphorylation, which depends on its nature/target may change the local conformation to favor its association with various different proteins. TNFR1 tyrosine phosphorylation has been reported, and phosphotyrosine residue within the YXXL/V sequence has been identified within the death domain of TNFR1 or other death receptors (5,6). Initial reports suggested that TNFR1 tyrosine phosphorylation had a negative effect on TNF-induced NF-kB activation and growth modulation (6,7). However, no consensus death domain recognition motif with phosphotyrosine has been established from sequence alignment or binding analysis among these death domain factors. Also, the significance of TNFR1 serine phosphorylation on death domain signaling remains elusive, despite the reports of TNFR1 and FADD serine phosphorylation (7)(8)(9).
In this work, we have analyzed TNFR1 and TRADD serine phosphorylation and investigated the role of the SXXE/D motifdependent TNF signal transduction. Conserved SXXE/D motifs have been identified in the death domains of TNFR1, TRADD, FADD, and RIP. We show in this study that the phospho-SXXE/D motifs of TNFR1 and TRADD death domains play a critical role in the death domain-death domain protein interaction and death domain signal transduction. TNF can also induce NF-kB activation or other cellular responses in a variety of cell types. We show in this study that TNF-induced and IkB kinase (IKK) b-dependent TNFR1 death domain S381 phosphorylation plays an essential role in TNFR1-TRADD interaction, leading to NF-kB activation, T cell proliferation, apoptosis, and migration. TNF plays a central role in the pathophysiology of inflammatory bowel disease (IBD) as well as rheumatoid arthritis. Anti-TNF therapy has been documented to have clinical efficacy in treating these inflammatory diseases. In this article, we provide evidence that in IBD, accumulating T cells in the inflamed intestinal tissues may do so in response to constitutive TNFR1 S381 phosphorylation in these inflammatory lymphocytes. Taken together, these findings provide insights into the determinants of specificity for death domain complex formation during signal transduction, which leads to inflammatory T cells activation, proliferation, and migration. Inasmuch, we suggest that SXXE/D motifs of death domain receptors or adaptors may provide novel targets for future therapeutic intervention.

T and B cells preparation and migration and immunostaining in situ
Human primary T cells from healthy blood donors were prepared by centrifugation of fresh blood through Histopaque of Sigma-Aldrich and taking the buffy coat. Cells were prepared by adsorbing the monocytes to gelatin-coated plates and culturing nonadherent lymphocytes in RPMI 1640 medium supplemented with 10% FBS and PHA (1 mg/ml) for 3 d, followed by culturing in 20 ng/ml IL-15 for 3∼6 d. Flow cytometric analysis demonstrated that these cells were .97% CD3 positive and CD56 negative. B cells were prepared from mouse spleen or human blood of healthy donors according to the published protocol (12). Flow cytometric analysis demonstrated that these enriched B cells were .97% positive for CD19 expression and lacked expression of CD4 and CD8. Real-time migration of human primary T cells on cover glass with ICAM-1 and with or without TNF (50∼200 ng/ml) treatment was studied by time-lapse video microscopy. Time-lapse movies were taken every 5 s. T cells were maintained at 37˚C with a FCS2 live cell imaging chamber (Bioptechs) in IL-15 (20 ng/ ml) contained glucose medium. Intestinal tissue samples obtained from non-IBD (normal), Crohn's disease, and ulcerative colitis were immunostained with anti-pS381-TNFR1, anti-CD3, and anti-CD20 according to the published protocol (13).
Coimmunoprecipitation, protein phosphorylation assay, DNA binding, luciferase reporter assay, and apoptosis analysis First, for coimmunoprecipitation, equal amount of cell lysates prepared in RIPA lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, and 0.5% Nonidet P-40) containing protease inhibitor mixture, were incubated with the protein A/G-agarose bead-conjugated Ab overnight at 4˚C. The Ab beads were extensively washed and suspended in Laemmli sample buffer (Bio-Rad) and boiled for 3-5 min. Such prepared immunoprecipitates or the wholecell lysates (5∼10 mg) were analyzed by loading into 10% SDS-PAGE for electrophoresis, transferred to nitrocellulose membrane, and followed by Western blotting analysis. Second, for TNFR1 or TRADD phosphorylation analysis in 293T cells, TNFR1 was transiently transfected in 293T cells for 24 h, followed by [ 32 P]orthophosphate metabolic labeling for an additional 6 h. Immunoprecipitated TNFR1 proteins were analyzed in 10% SDS-PAGE. Radiolabeled proteins transferred to the nitrocellulose membrane were exposed to x-ray film for autoradiography. TNFR1 expression level was analyzed with anti-TNFR1 blot. For TNFR1 phosphorylation in vitro, immunopurified Flag-IKKa, Flag-IKKb, or Flag-RIP (500 ng) was incubated in kinase reaction buffer containing 10 mCi g-[ 32 P]ATP, 1 mM ATP, 10 mM MgCl 2 , 1 mM DTT, 100 mM NaCl, and 50 mM Tris-HCl (pH 7.8) at 30˚C for 15 min. The substrate in these in vitro reactions was GST-TNFR1 (1 mg). In a separate experiment, GST-TNFR1 wild-type (WT) or GST-TNFR1-S381A was incubated with purified Flag-IKKb in kinase reaction buffer without isotope and TNFR1 phosphorylation was examined by blotting with anti-pS381-TNFR1. Third, for DNA binding analysis, nuclear fractions were incubated with kB-site DNA oligo-beads overnight at 4˚C. After extensive washes, the DNA beads binding NF-kB were analyzed in Western blot with anti-Rel A. Fourth, for luciferase activity analysis, 293T cells (1 3 10 5 ) were transfected with Lipofectamine 2000. A dual-luciferase reporter assay system was applied according to the instructions of the manufacturer (Promega). pRL vector (30 ng) was transfected along with 2xkB-luciferase reporter (300 ng) and 2 mg of either empty vector or TNFR1 or TRADD in WT or mutant forms. Firefly luciferase activity was measured in a luminometer and normalized on the basis of Renilla luciferase activity. Fifth, for cell viability analysis, cells were either stained with propidium iodide (PI) (0.5 ml PI staining reagent: 0.1% Nonidet P-40, 0.1% sodium citrate, and 50 mg/ml PI; Sigma-Aldrich) for flow cytometric analysis or stained with trypan blue for exclusion counting (14).

TNF induced T cell morphological change is serine phosphorylation dependent
T cells but not B cells responded to TNF stimulation by a morphological change during cell migration (Fig. 1A). However, this morphological change in T cells was inhibited by treatment of serine kinase inhibitor H7 (Fig. 1A). TNF-induced T cell numbers with morphological changes were reduced by treatment of staurosporine (Sigma-Aldrich), another serine kinase inhibitor, to a lesser extent when compared with H7 (Fig. 1B). These data indicated that T cell morphological change induced by TNF was regulated by serine phosphorylation.

TNFR1 and TRADD death domains are phosphorylated on the SXXE/D motif
Secondary structure-based alignment indicates that TNFR1, TRADD, FADD, and RIP bear YXXL/V motifs and/or SXXE/D motifs in different a-helical segments of their death domains ( Fig. 2A). TNFR1 death domain carries two YXXV/L motifs (Y360AVV and Y401SML) and one SXXE motif (S381DHE) conserved in all species (5)(6)(7). Y401XXL motif of a-helix4 is conserved on all death receptors as well as on RIP whose phosphorylation has been reported to be necessary for recruiting the SH2 domain bearing tyrosine phosphatase SHP-1 (6).
In 293T cells, although WT TNFR1 was shown to recruit the SH2 domain bearing SOCS3, TNFR1 with Y401→F substitution attenuated TNFR1-SOCS3 interaction (data not shown). To determine the importance of these YXXL/V motifs in TNFR1 death domain signaling, we examined TNFR1 and TRADD interaction, in which single or doubly Y→F mutated TNFR1 (i.e., TNFR1-Y360F, TNFR1-Y401F, and TNFR1-Y360F/Y401F) were produced. These mutants showed no apparent defect either in TNFR1-TRADD interaction or in their capacity to induce apoptosis (Supplemental Figs. 1, 2), even though partially inhibiting NF-kB activation (Fig. 2B). By contrast, TNFR1 with S381→A substitution (TNFR1-S381A) inhibited both NF-kB activation and apoptosis induction significantly (Fig. 2B, Supplemental Fig. 3). With E384→A substitution, TNFR1-E384A also affected NF-kB activation (Supplemental Fig. 3), suggesting SXXE/D motif as a whole is important for phospho-SXXE/D motif modulatory signaling. These data suggest a more prominent role of serine phosphorylation than tyrosine phosphorylation in TNFR1 death domaindependent intracellular signal transduction. TNF-induced T cell morphological change is serine phosphorylation dependent. A, Peripheral T cells and peripheral B cells obtained from healthy blood donors were cultured in the chambers coated with ICAM-1 in the presence or absence of TNF (200 ng/ml) with or without serine kinase inhibitor H7 pretreatment for 1 h and immediately subjected to time-lapse fluorescence imaging at 37˚C. Cell morphological changes were recorded at time 0 or 30 min after. Representative differential interference contrast microscope images show a randomly selected region at five-fold magnification. B, Peripheral T cells and peripheral B cells obtained from healthy blood donors were cultured in the chambers coated with ICAM-1 in the presence or absence of TNF (50 ng/ml) with or without a different dose of serine kinase inhibitors H7 or staurosporine pretreatment for 1 h and immediately subjected to timelapse fluorescence imaging at 37˚C. Cell numbers with morphological changes were counted after 30 min. Data presented are mean 6 SD of triplicate determinations. The death domains of TNFR1 and TRADD bear phospho-SXXE/D motifs. A, The death domains of TNFR1, TRADD, FADD, and RIP from different species were analyzed with the three-dimensional PSSM web tool version 2.5.6 (http://www.sbg.bio.ic.ac.uk/~3dpssm/) for a-helix and b-sheet prediction. B, Empty vector (EV), WT, TNFR1, TNFR1-Y360F, TNFR1-Y401F, or TNFR1-S381A was cotransfected with 2xkB-luciferase reporter in 293T cells for 24 h, followed by luciferase reporter activity measurement. Data presented are mean 6 SD of triplicate determinations. One representative anti-TNFR1 Western blot of indicated TNFR1 forms in 293T transfectants was shown in lower panel. C, EV or an indicated form of TNFR1 was transiently transfected in 293T cells, followed by [ 32 P]orthophosphate metabolic labeling. Immunoprecipitated TNFR1 proteins transferred to the nitrocellulose membrane were exposed to x-ray film for autoradiography. TNFR1 expression level was analyzed with anti-TNFR1 blot. Lower panel, EV or TNFR1 transiently transfected 293T cells were treated with TNF for 30 min, followed by Western blot analysis with anti-pS381-TNFR1 or anti-TNFR1. D, Wholecell extracts, prepared from HeLa cells treated with TNF for indicated times, were subjected to Western blot analysis with anti-pS381-TNFR1, anti-IkBa, or anti-TNFR1. E, TRADD of indicated forms were transiently transfected in 293T cells for 24 h, followed by [ 32 P]orthophosphate metabolic labeling for additional 6 h. The autoradiography of the immunoprecipitated TRADD were analyzed as described above. TRADD expression level was analyzed with anti-TRADD blot.
We subsequently examined phosphorylation of the TNFR1 death domain SXXE motif in greater detail. TNFR1-S381A mutant or TNFR1 with "S381DHE" motif deletion mutation (TNFR1-DS381XXE) largely abolished TNFR1 phosphorylation in 293T cells, demonstrating that TNFR1 mainly undergoes serine phosphorylation rather than tyrosine phosphorylation (Fig. 2C). To specifically detect TNFR1 phosphorylation on S381, we prepared a polyclonal Ab against phospho-S381 of TNFR1. With this Ab, TNF-dependent TNFR1 phosphorylation on S381 was clearly detected in 293T cells (Fig. 2C, lower panel) or in HeLa cells (Fig. 2D). It is worth noting that when overexpressed in 293T cells, TNFR1 became constitutively phosphorylated on S381 (Fig.  2C, lower panel).

The phospho-SXXE/D motif intermediates the death domain modular interaction
To investigate the role of phospho-SXXE/D motifs in death domain-death domain protein interaction, TRADD was cotrans-fected with WT TNFR1 (TNFR1-WT) or TNFR1 mutant (i.e., TNFR1-S381A or TNFR1-DS381XXE) in 293T cells. TNFR1-TRADD interaction was inhibited by TNFR1 with S381A mutation and worsened by TNFR1 with S381XXE deletion as revealed by coimmunoprecipitation analysis (Fig. 3A). We also examined TNFR1-TRADD complex stability by carrying out a peptide competition experiment, in which the synthesized unphosphorylated or phosphorylated peptide containing S381XXE motif (position 375-395) was incubated with the immunopurified TNFR1-TRADD complex, respectively. At a concentration of 300 nM, the phospho-peptide but not the control peptide disrupted the TNFR1-TRADD complex, indicating that phospho-S381XXE motif of TNFR1 death domain is involved in the formation of a stable TNFR1-TRADD complex (Supplemental Fig. 5).
We next sought to identify the protein kinase responsible for TNFR1 phosphorylation on S381. Coimmunoprecipitation analysis revealed that both IKKa and IKKb could form a complex with TNFR1 in 293T cells with IKKa or IKKb transfected along with TNFR1 ( Supplemental Figs. 6, 7). Bacteria-purified GST-TNFR1 was applied to an in vitro kinase assay in the presence of g-[ 32 P] ATP, revealing a synergistic effect between IKKa and IKKb on TNFR1 phosphorylation; despite that, IKKb alone was more efficient than IKKa in TNFR1 phosphorylation induction (Fig. 3B). When purified TNFR1, WT but not S381A form, was incubated with purified Flag-IKKb, in an in vitro reaction, TNFR1-S381 phosphorylation was clearly detected with the Ab against pS381-TNFR1 (Fig. 3C). This supports the conclusion that IKKb is able to phosphorylate TNFR1 on S381 in 293T cells (Supplemental Fig. 8). However, in IKKb 2/2 mouse embryonic fibroblasts (MEFs), there was a basal level of TNFR1 S381 phosphorylation (Fig.  3D), which was most likely contributed by endogenous IKKa. Importantly, TNFR1-TRADD complex formation was induced by FIGURE 3. The phospho-SXXE/D motif intermediates the death domain modular interaction. A, In 293T cells, TNFR1 of indicated forms were cotransfected with TRADD, followed by coimmunoprecipitation analysis. Anti-TNFR1 immunoprecipitates were subjected to Western blot analysis with anti-TRADD or anti-TNFR1. B, Flag-tagged IKKa, IKKb, and RIP were immunoprecipitated from 293T transfectants and were incubated with GST-TNFR1 purified from bacteria in phosphorylation reaction buffer containing g-[ 32 P]ATP for 15 min at 30˚C. Radiolabeled proteins were separated in 10% SDS-PAGE, transferred to the nitrocellulose membrane, and exposed to x-ray film for autoradiography. C, Purified GST-TNFR1 was incubated with purified Flag-IKKb in reaction buffer, followed by Western blot analysis with anti-pS381-TNFR1 or indicated Abs. D, Whole-cell extracts, prepared from WT or IKKb 2/2 MEFs treated with TNF for indicated times, were subjected to Western blot analysis with anti-pS381-TNFR1 or anti-TNFR1. E, Whole-cell extracts were prepared from WT or IKKb-deficient MEFs treated with or without TNF (50 ng/ml) for 15 min. Anti-TNFR1 immunoprecipitates were analyzed with anti-TRADD in a Western blot. F-H, In 293T cells, TRADD of indicated forms were transfected along with TNFR1 (F), FADD (G), or RIP (H), followed by coimmunoprecipitation with anti-TNFR1 (F) or anti-TRADD (G, H) and blotted with indicated Abs in Western blot analysis. TNF treatment of WT MEFs but not in IKKb 2/2 MEFs (Fig. 3E), suggesting that IKKb may play an important role in both TNFR1 and TRADD activation.
Because TNFR1-TRADD complex formation was not completely abolished when TNFR1 was introduced with S381A mutation or S381XXE deletion mutation (Fig. 3A), we suspected that TRADD serine phosphorylation was also involved in TNFR1-TRADD complex formation. TNFR1-TRADD complex formation was largely abolished when both S215A and S296A mutations were introduced (Fig. 3F), suggesting that both pS215XXD and pS296XXE motifs of TRADD were involved in TNFR1-TRADD interaction (2,15). Moreover, TRADD phosphorylation on S215XXD and S296XXE motifs performed an important role in TRADD-FADD or TRADD-RIP complex formation, because TRADD-S215A/S216A but not TRADD-S215A or TRADD-S296A failed to recruit FADD or RIP in 293T cells (Fig. 3G, 3H). These results strongly indicate that the two phospho-SXXE/D motifs of the TRADD death domain play a central role in this death domain-tetramer complex formation.

NF-kB-dependent gene regulation requires the SXXE/D motif phosphorylation
Given that TNFR1-TRADD complex formation led to IkB degradation and NF-kB nuclear translocation, a potentiating effect of the SXXE/D motif phosphorylation in their death domains was expected to initiate NF-kB activation. In 293T cells, transient transfection of either TNFR1 or TRADD alone could trigger NF-kB transcriptional activation, as reflected by the results of NF-kBdependent luciferase reporter activation assays of Fig. 4A and 4B. Although TNFR1-S381A, TRADD-S215A, and TRADD-S296A mutants reduced NF-kB activation, TNFR1-DS381XXE, TNFR1-E384A, and TRADD-S215A/S296A mutants almost completely abrogated NF-kB activation (Fig. 4A, 4B). Along these lines, we found that TRADD-dependent NF-kB activation was further enhanced by cotransfecting TRADD with TNFR1-WT but not with TNFR1 mutants (Fig. 4C). These results strongly support our model that both the phosphorylated serine and the negatively charged p+1 residue of the "SXXE/D motif" in the death domains are important in NF-kB activation.

TNF activates NF-kB in T cells via TNFR1 S381 phosphorylation and TNFR1-TRADD complex formation
It has been reported that both T and B cells respond to TNF for activation during inflammatory processes. However, TNF treatment for 10 or 30 min induced TNFR1 S381 phosphorylation in Jurkat T lymphoma line and mouse thymocytes that were primarily T cell but not in Daudi cells, a B lymphoma line, or mouse splenocytes that contained 40% B cells (Fig. 5A). TNF treatment of Jurkat cells induced the formation of a complex between TNFR1 and TRADD (Supplemental Fig. 9), supporting the conclusion that serine phosphorylated TNFR1 and serine phosphorylated TRADD form modulatory protein-protein interaction upon TNF treatment. TNF treatment induced IkB degradation and NF-kB activation for DNA binding in Jurkat cells and mouse thymocytes but not in Daudi cells, mixed mouse splenocytes, or purified mouse spleen B cells (Fig. 5B). When another pair of human T and B lymphoma lines, H9 and SKW6.4, or purified peripheral T and B cells from normal human blood donors were tested, IkB (IkBa) degradation and NF-kB (Rel A) nuclear translocation were only detected in TNF-treated T cells and not in TNF-treated B cells (Fig. 5C, 5D).
The significantly lower expression level of TNFR1 by B cells has been suggested as an explanation for the failure of B cells to response to TNF treatment by NF-kB activation (Fig. 5A, 5C). However, TNFR1-associated death domain factors including TRADD, FADD, and RIP are all expressed at comparable levels in both B and T cells, whereas TNFR2 expression level was low overall in both T and B cells (data not shown). The TNFR1associated inhibitor A20 has been reported to induce RIP polyubiquitination and inhibit TRADD downstream signal transduction in T cells (15,16). With mass spectrometry for proteomic analysis, no apparent difference was detected for proteins associated with TNFR1 in B cells with or without treatment of A20 or other established inhibitors (data not shown). LPS induced IkB degradation in both thymocytes and splenocytes (Fig. 5E), suggesting that a similar level of IKK activity is present in T and B cells.
We then examined the effect of TNF stimulation on T and B cell viability. TNF treatment for 12-24 h stimulated proliferation in peripheral T cells and Jurkat cells, but not in Daudi cells (Fig. 5F). We also compared different sources of T and B cells for their apoptotic response to TNF treatment. In this regard, we found that TNF-mediated apoptosis could be detected in Jurkat cells or thymocytes, whereas only a spontaneous or background level of apoptosis was observed in Daudi cells or splenocytes (Supplemental Fig. 10). These results indicate that TNF can selectively activate T cells over B cells via NF-kB activation, and this appears to require serine phosphorylation-dependent TNFR1-TRADD complex formation. . NF-kB-dependent gene regulation requires the SXXE/D motif phosphorylation. A, EV and different forms of TNFR1 were transiently transfected with 2xkB-site luciferase reporter in 293T cells for 24 h, followed by luciferase reporter activity measurement. Data presented are mean 6 SD of triplicate determinations. Experiments were repeated three times. One representative anti-TNFR1 Western blot of indicated TNFR1 forms in 293T transfectants was shown in the lower panel. B, EV and different forms of TRADD were transiently transfected with 2xkB-site luciferase reporter in 293T cells for 24 h, followed by luciferase reporter activity measurement and data process as described in A. C, EV or TRADD WT was transfected with 2xkB-site luciferase reporter alone or with different forms of TNFR1 cotransfection in 293T cells for 24 h, followed by luciferase reporter activity measurement and data process as described in A.

TNF induces T cells migration via TNFR1 S381 phosphorylation
Among TNF's variety of actions are the regulation of cell viability and the induction of cell migration. To investigate the role of TNFR1 in regulating cell migration, fibroblasts obtained from WT, TNFR1 2/2 , TNFR2 2/2 , and TNFR1/TNFR2 double 2/2 mice were assessed for their comparative ability to migrate using a cell chamber migration assay. Interestingly, although TNFR2 has been previously shown to play a more critical role than TNFR1 in cell migration (17), we observed in this study that neither TNFR1 2/2 alone nor TNFR2 2/2 alone showed any negative effect on cell migration (Fig. 6A). However, cell migration was severely inhibited when both TNFR1 and TNFR2 were knockout (Fig. 6A), suggesting that TNFR1 and TNFR2 play a collaborative role in regulating cell migration. Furthermore, reconstitution of TNFR1-WT but not TNFR1-S381A mutant form into TNFR1/TNFR2 2/2 partially restored cell migration (Fig. 6A), indicating that TNFR1 involvement in cell migration is mediated through S381 phosphorylation. We then performed RT-PCR to examine the expression of genes related to cell migration in these TNFR1 2/2 or TNFR1,TNFR2 2/2 MEFs. Among those genes assessed, IL-6, IL-8, matrix metallopeptidase-9, and cyclin D1 were apparently reduced in their expression in TNFR 2/2 MEFs when compared with WT MEFs (Fig. 6B). The introduction of TNFR1-S381A mutant in these TNFR1/TNFR2 2/2 MEFs showed little or no rescue effect on regulation of the expression of those genes (Fig.  6B). Thus, we believe that TNFR1 phosphorylation on S381 plays a critical role in NF-kB-dependent gene regulation involved in cell migration and that the presence of both TNFR1 and TNFR2 on the cell is still critical for cell migration.
We then compared T cell and B cell migration in response to TNF treatment. In the absence of TNF, T cells prepared from peripheral blood of normal donors, remained still in their ICAM-1-coated chamber without evidence of observable crawling or movement in a directed fashion (Fig. 6C). In the presence of TNF, T cells became polarized and migrated on the ICAM-1-coated surface with an approximate steady state migration velocity of 10 mm/min (Fig. 6D). These migrating T cells exhibited rapid shape change, formation of constriction rings, and concomitant cytoplasmic streaming. The migrating T cells treated with TNF were comparable with those exposed to treatment for integrin activation (18). Under the same condition, peripheral B cells failed to respond to TNF treatment with respect to the development of directed migration or migration related morphological change (Fig. 6C, 6D). Pretreatment of T cells with IKKb inhibitor PS1145 abolished T cell migration in response to TNF stimulation (Fig.  6E). Above results demonstrated that TNFR1 S381 phosphorylation by IKKb leads to NF-kB activation and downstream gene expression is required for TNF to induce a T cell migration response.

T cells with constitutive expression of TNFR1-S381 phosphorylation accumulate in the inflamed intestines of IBD patients
InIBD, TNF plays an essential role in recruiting immune cells to inflammatory sites (19). Intestinal tissue samples obtained from patients of Crohn's disease or ulcerative colitis were compared with normal intestinal tissues for T cell accumulation. In all the intestinal tissue samples analyzed, T cells became most apparently in the inflamed mucosa in both Crohn's disease and ulcerative colitis specimens (Fig. 7A). These accumulating T cells were exclusively phospho-S381-TNFR1 positive (Fig. 7A). In contrast, B cells, localized mainly in the intestinal B cell follicles or in the germinal centers, were sparsely visible (Fig. 7B). These results suggest that TNFR1 S381 constitutive phosphorylation is likely involved in activating and/or driving the influx of T cells into the areas of inflammation seen in the bowel (19,20). Further supporting the requirement of SXXE/D motif phosphorylation during signal transduction via TNFR1-TRADD-centered death domain complex formation.

Discussion
Like tyrosine phosphorylation, serine phosphorylation plays a critical role in regulating signaling for cell survival, apoptosis, or cell migration (21,22). SXXE/D motifs, found in the cytoplasmic domains of many TNFR family members as well as their adaptor proteins, are involved in serine signal transduction. However, unlike PXSP or PS/SP motifs that are phosphorylated by MAPK/ cdc2PK type kinases for b-stranded WW domain (bb) interaction (23), SXXE/D motifs are phosphorylated by CK-II/IKK type kinases and have been shown to associate with TNFR1 (24). We demonstrated here that IKKb and, to a lesser extent, IKKa can phosphorylate the "SXXE" motif of TNFR1 death domain.
In the helical death domain (aaaaaa) of Fas, the a-helix3 is involved in direct contact with FADD death domain within the a-helix2-a-helix3 region, which bears the SXXE/D motif (25). Besides, S215 of TRADD death domain and positive charge residues of both TRADD and TNFR1 death domains have been previously noted to play a role in the interaction between these two molecules (26,27). Both SXXE/D motifs of the TRADD death domain were required for upstream TNFR1 and downstream FADD/RIP binding simultaneously, suggesting that TRADD may either form dimer prior to mediating the TNFR1 and FADD/RIP complex formation or sequentially recruit TNFR1, FADD, and RIP. Furthermore, the TNFR1 SXXE motif has also been demonstrated to be important for TNFR1-TRADD complex formation. . TNF induces T but not B cell migration. A, Equal amount (5 3 10 5 ) of mouse embryonic fibroblasts obtained from TNFR1 2/2 , TNFR2 2/2 , or TNFR1 and TNFR2 double 2/2 (TNFR1,2 2/2 ) mice, as well as the TNFR1-WT or TNFR1-S381A reconstituted fibroblasts were seeded in the upper chamber (polycarbonate membranes with 8.0-mm pore size) in DMEM supplied with 5% FBS. Twelve hours later, the cells migrated to the low chamber were fixed, stained with H&E, and visualized with a light microscope according to our published protocol (13). The above H&E stained cells in the lower chamber were counted from three vision areas with a light microscope. B, In TNFR1 2/2 MEFs or TNFR1,TNFR2 2/2 MEFs with TNFR1-S381A mutant reintroduction, indicated genes were analyzed for their transcriptional regulation by performing RT-PCR. Data are expressed as mean 6 SD of three experiments. C, Peripheral T cells and peripheral B cells obtained from healthy blood donors were cultured in the chambers coated with ICAM-1, in the presence or absence of TNF (200 ng/ml) and immediately subjected to time-lapse fluorescence imaging at 37˚C. Cell morphological changes were recorded at different times (min) as indicated. Experiments were repeated on T lymphocyte preparations from three independent donors. D, The migration of the above T and B cells in C was tracked over a 30-min period. Each line represents one cell. E, TNF treatment at indicated time failed to induce cell morphological change in peripheral T cells pretreated with IKKb inhibitor PS-1145 for 2 h. Representative differential interference contrast microscope images show a randomly selected region at five-fold magnification (C, E).
These observations are reminiscent of those made for the a-helix sandwiched and b-strand-centered SH2 domains of Src (abbbba) and STAT (abbbaa) that bear phosphorylated tyrosine residues (28,29). For two STAT protein molecules, positively charged residues of aA and bB of their SH2 domains recognize reciprocal phospho-tyrosine residue located at the extended C terminus of the SH2 domain during dimerization (28). Serine phosphorylation of the SXXE/D motif is important for death domain-mediated modular interaction. S381 phosphorylation of TNFR1 death domain SXXE motif may enhance the proteinprotein interaction by juxtaposing another negatively charged group adjacent to D382 and E384 for stabilizing its association with the positive groove in TRADD. Comparing phosphotyrosine with, phosphoserine indicates phosphoserine is less bulky in structure. Thus, the negatively charged residue at the p+3 position in the death domain SXXE/D motif presumably plays a more important role in strengthening the modular interaction than the hydrophobic residue of YXXL//I/V/M/Q motif does in interacting with the SH2 domain.
The effect of the phospho-SXXE/D motif may not only be limited to the homologous domain interaction. TNFR2 bears at least three species-conserved SXXE/D motifs within the C terminus. The helical ankyrin repeats (ANK) of ASB3 and the TRAF-C domain of TRAF2 were found to dock on those TNFR2 SXXE motifs that are most likely phosphorylated (10,30). Phospho-SXXE/D motifs have also been identified in other helical structures such as ANK of IkBa. Ubiquitin-protein ligases are responsible for phospho-SXXE/D recognition during IkB degradation and the release of NF-kB. Positively charged residue of R47 within the helical ANK of p16INK4a interacts with a negatively charged residue (i.e., Glu) of the N-terminal SXXE motif of cdk4 (31).
The observation that TNF preferentially activates T cells via SXXE motif phosphorylation within TNFR1 death domain, and this signaling may explain why T cells but not B cells accumulated in areas of inflammation, thus playing a dominant role in inflammatory process. Although TNF may activate other signaling pathways such as JNK in both T and B cells, SXXE and/or SXXD motif-mediated NF-kB activation appears to play a more critical role in cell growth regulation, apoptosis, cell migration, and the accumulation of T cell at sites of inflammation (11,32). It is tempting to think that TNFR1-S381 may become constitutively phosphorylated in T cells in inflamed intestinal mucosa, presumably because of increased TNF level in the diseased lamina propria as opposed to the adjacent uninflamed bowel. Furthermore, we suggest that constitutive phosphorylation of TNFR1 should promote inflammation by regulating T cells proliferation and migration to the inflamed gut of IBD patients (33). Thus, we feel that the disruption of the modular death domain-death domain interaction in T cells, by targeting phospho-SXXE/D motifs, may represent a potentially novel therapeutic approach for the treatment of inflammatory conditions, like IBD. FIGURE 7. T cells accumulated in the inflammatory intestines of IBD patients are TNFR1-S381 constitutively phosphorylated. A, Local intestinal mucosa from control non-IBD, inflamed Crohn's disease, or inflamed ulcerative colitis were immunostained with anti-CD3, anti-pS381-TNFR1, and anti-CD20, respectively. B, Intestinal tissue samples obtained from non-IBD, Crohn's disease, and ulcerative colitis were immunostained with anti-CD20 to show the B cell follicles.