| p38 is a subfamily of the mitogen-activated protein kinase (MAPK) superfamily, which also includes extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK). p38 activation depends on the phosphorylation of Thr180 and Tyr182 in the Thr-Gly-Tyr motif of the activation loop, which is typically mediated by sequential protein phosphorylation through a MAPK module: namely, MAPK kinase kinase (MAK3K)→MAPK kinase (MAP2K)→MAPK. Physical stresses such as UV and proinflammatory cytokines such as tumor necrosis factorα(TNF-α) activate the classical p38 activation pathway. MAPK kinase 3 (MKK3) and MKK6 are the principal MAP2Ks responsible for the dual phosphorylation of p38 in the classical activation pathway. Besides, alternative mechanisms of p38 activation based on intramolecular autophosphorylation at Thr180 and Tyr182 have been reported.p38 is usually considered to be a central regulator of inflammation. However, enormous evidence including our recent work shows that p38 also plays a key role in modulating cell death. p38 activation mediated by both sequential protein phosphorylation and autophosphorylation seems to be involved in the regulation of cell death. For example, several previous studies of our group have revealed that p38 plays a pro-survival role in TNF-α-induced cell death in fibroblasts and L929 fibroblastoma cells by contributing to the full activation of nuclear factor-κB (NF-κB). Conversely, p38αautophosphorylation upon binding to TAB1 [transforming growth factor-β(TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1] results in augmented cell death during myocardial ischemia reperfusion. Despite of the clarification of the pivotal roles of the p38 pathway in determining cell fate, the detailed molecular mechanisms of p38 activation in the two processes remain unclear. Adaptor protein TAB1 can directly interact with p38α, but not with the other p38 isoforms. The unique interaction between TAB1 and p38αmakes it a potential therapeutic target for reperfusion injury. Thus, it is of importance to disclose the precise three dimensional (3-D) structure of p38α/TAB1 complex. On the other hand, even though adaptor proteins have been demonstrated indispensable for the sequential protein phosphorylation of the ERK pathway and the JNK pathway, surprisingly few adaptor proteins have been identified for the classical p38 pathway in mammalian cells. Our previous work has demonstrated that GNB2L1, an adaptor protein that contains seven Trp-Asp (WD) repeats and has been implicated in the regulation of multiple signaling pathways, contributes to TNF-α-induced JNK activation via directly binding with MKK7. Therefore, it is of interest to investigate the roles of GNB2L1 in TNF-α-induced p38 activation and TNFα-induced cell death.In this study, with the combination of computer-guided homology modeling, ab initio modeling, molecular docking and traditional cell biology methods, we identified key amino acids essential for the specific interaction between TAB1 and p38α. Asp230 and Tyr258, as well as Thr218, in p38αwere determined to be critical for TAB1 interaction. Whereas the corresponding key sites in TAB1 were Ser399, Ser401, Asn436 and Thr440. Mutation of these key sites led to decreased or enhanced binding between TAB1 and p38α, as suggested by the theoretical 3-D modeling complex structure. However, TAB1-dependent p38αphosphorylation dramatically decreased in either case, suggesting only the most appropriate binding of TAB1 and p38αled to optimal p38αautophosphorylation. Based on these findings, several antagonistic peptides were designed to interrupt the p38α/TAB1 interaction. Among the antagonistic peptides that could block the p38α/TAB1 interaction in vitro and in vivo, one (PT5) was synthesized as the D-retro-inverso form (D-PT5) to prevent degradation. The D-retro-inverso decapeptide HIV-TAT [48-57] transporter sequence was added at the carboxy-terminal to convey cell permeability, separated from the antagonistic peptide by the insertion of di-D-proline. D-PT5 inhibited the interaction between p38αand TAB1 and inhibited TAB1-dependent p38αphosphorylation in vivo. Furthermore, D-PT5 significantly alleviated the rat myocardial injury caused by ischemia reperfusion. Collectively, our work might contribute to the production of selective inhibitors targeting p38α/TAB1 interaction for the prevention or treatment of myocardial injury caused by ischemia reperfusion.Our data also show that GNB2L1 could directly interact with MKK6 in vitro and in vivo. WD1 of GNB2L1 was required for the specific interaction between GNB2L1 and MKK6. GNB2L1 overexpression led to enhanced p38 activation and rendered L929 cells resistant to TNF-α-induced cell death, whereas GNB2L1 knock down exhibited opposite effects. GNB2L1ΔWD1 showed reduced ability to enhance p38 activation. Thus, our work has identified a novel adaptor protein essential for the classical p38 pthway.
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