The Molecular Mechanism Of Interaction Between Neurotrophins Receptor TrkA And Eukaryotic Translation Initiation Factor eIF4A

Neurotrophins are a family of closely related proteins that were identified initially as survival factors for sensory and sympathetic neurons, and have since been shown to control many aspects of survival, development and function of neurons in both the peripheral and the central nervous systems. Four neurotrophins are expressed in mammals:NGF, BDNF, neurotrophin-3(NT-3) and neurotrophin-4/5(NT-4/5). The biological effects of neurotrophins require tropomyosin-related kinase (Trk) receptors and p75NTR receptors. Each of the four mammalian neurotrophins has been shown to activate one or more of the three members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB and TrkC). In addition, each neurotrophin activates p75neurotrophin receptor (p75NTR). Though Trk receptors, neurotrophins activate Ras, phosphatidyl inositol-3(PI3)-kinase, phospholipase C-yl and signalling pathways controlled though these proteins, such as the MAP kinases.Eukaryotic translation initiation factor4F(eIF4F) is a protein complex that mediates recruitment of ribosome to mRNA. eIF4F is a complex of thee polypeptides:eIF4A, an RNA-dependent ATPase and RNA helicase; eIF4E, a cap-binding protein; eIF4G, a scaffold protein. eIF4A is an ATP-dependent RNA helicase that functions in translation initiation to catalyze the unwinding of mRNA secondary structure at the5’UTR. There are thee mammalian isoforms:eIF4A1, eIF4A2, eIF4A3. eIF4A is a target of Pdcd4, Patemine A,15d-PGJ2, Hippuristanol, BC1RNA which can block translation initiation.In our previous study, we demonstrated RanBPM could interact with TrkA and p75NTR simultaneously and form a ternary protein complex though co-immunoprecipitation assay, at the same time, some other protein, such as eIF4A1, also existed in the TrkA-RanBPM-p75NTR immunocomplex which identified by RPLC-MS/MS measure. Our research focuss on molecular mechanism of interaction between neurotrophins receptor TrkA and eukaryotic translation initiation factor eIF4A.In this study, we found1. TrkAICD could interact with eIF4Al and the interaction was confirmed by yeast two-hybrid assays, GST pull-down assays and co-immunoprecipitaiton studies.2. TrkAICD-eIF4A1association though N terminal of eIF4A1was confirmed by GST pull-down assays, and the region of TrkAICD interacted with the eIF4A1was located in the tyrosine kinase domain.3. TrkA could interact with eIF4A1and the interaction was confirmed by GST pull-down assays and co-immunoprecipitaiton studies.4. In vivo translation assays showed that TrkA could promote translation initiation though association with eIF4A1.5. In vivo ubiquitylation assay showed that eIF4A1could reduce TrkA receptor ubiquitination. Recently, a novel group of IFNs was discovered and named IFN-λ. The Nomenclature Committee of the International Society for Interferon and Cytokine Research has designated these novel cytokines type III IFNs. The new IFN family has thee members, named IFN-λ1, IFN-λ2, and IFN-λ3, or interleukin-29(IL-29)(λ1) and IL-28A/B (λ2/3). IFN-Xs act though IFN-λR, a class II cytokine receptor complex comprising CRF2-12(IFN-λR1) chain, which is specific for the IFN-λs, and the CRF2-4chain, which is also part of the receptors for IL-10, IL-22and IL-26. Both receptor chains are constitutively expressed on a wide variety of human cell lines and tissues and signal though the JAK-STAT pathway. This receptor-ligand system may contribute to antiviral or other defenses by a mechanism similar to type I IFNs.TNF receptor-associated factor (TRAF) family members are intracellular proteins that transmit signals from the intracellular portion of TNFR superfamily members. Currently, there are seven known mammalian TRAFs (TRAF1-TRAF7). TRAF6is unique among TRAF family members with regard to the manner in which it interacts with receptors. Two groups independently cloned TRAF6. One searched a DNA database for TRAF2-like sequences followed by cDNA library screening, and the other performed a yeast two-hybrid screening using CD40as bait. TRAF6is a crucial adapter molecule common to the IL-1R/TLR family and TNFR superfamily, regulating a diverse array of physiological processes, including adaptive immunity, innate immunity, bone metabolism.In our previous study, IFN-λR1ICD(the intracullar domain of IFN-λR1) and TRAF6could directly bind in GST pull-down assay and could be co-immunoprecipitated when exogenously expressed in HEK293T cells. To determine the interaction between IFN-λR1and TRAF6, we conducted exogenous co-immunoprecipitation and semiendogenous co-immunoprecipitation experiments. To further explore the function of IFN-λR1-TRAF6association and the related signal transduction pathway, we conducted NF-κB Promoter Assay and In vivo ubiquitylation assays.In our study, we found1. The interaction between IFN-λR1and TRAF6was confirmed by exogenous co-immunoprecipitation and semiendogenous co-immunoprecipitation assays.2. IFN-λ1could inhibit TRAF6-induced NF-κB activiaton.3. IFN-λR1could reduce autoubiquitylation of TRAF6.