Like tyrosine phosphorylation, serine phosphorylation plays a critical role in regulating signaling for cell survival, apoptosis, or cell migration (21
). 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 β-stranded WW domain (ββ) 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 IKKβ and, to a lesser extent, IKKα can phosphorylate the “SXXE” motif of TNFR1 death domain.
In the helical death domain (αααααα) of Fas, the α-helix3 is involved in direct contact with FADD death domain within theα-helix2-α-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
). 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.
These observations are reminiscent of those made for theα-helix sandwiched and β-strand centered SH2 domains of Src (αββββα) and STAT (αβββαα) that bear phosphorylated tyrosine residues (28
). For two STAT protein molecules, positively charged residues of αA and βB 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 protein-protein 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 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
). Phospho-SXXE/D motifs have also been identified in other helical structures such as ANK of IκBα. Ubiquitin-protein ligases are responsible for phospho-SXXE/D recognition during IκB degradation and the release of NF-κB. Positively charged residue of R47 within the helical ANK repeats of p16INK4α 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-κB 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
). It is tempting to think that TNFR1-S381 may become constitutively phosphorylated in T cells in inflamed intestinal mucosa; presumably due to increased TNF level in the diseased lamina propria as opposed to adjacent un-inflamed bowel. Further 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.