| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 94 === Latent membrane protein 1 (LMP1), which is an Epstein-Barr virus (EBV)-encoded oncoprotein, induces NF-kappaB signaling by mimicking the tumor necrosis factor receptor (TNFR). Unlike TNFR-based signaling, however, LMP1 signals primarily from intracellular compartments in a ligand-independent manner. In this study, we seek to further elucidate the mechanism underlying ligand-independent LMP1 signaling by exploring the cellular factors mediating the intracellular trafficking of LMP1. Using a yeast-two-hybrid screen, we identify a new LMP1-interacting molecule, prenylated Rab acceptor 1 (PRA1), which is involved in intracellular LMP1 trafficking and LMP1-induced NF-kappaB signaling. Co-precipitation analyses demonstrated that LMP1 interacts with PRA1 for the first time through LMP1’s transmembrane domain, in particular the membrane-spanning segments 3-6. The fluorescence resonance energy transfer (FRET) microcopy and advanced bioluminescence resonance energy transfer (BRET2) assay revealed that LMP1 specifically interacts with PRA1 at the Golgi apparatus in living cells. The immunofluorescence microscopy further showed that co-localization of LMP1 and PRA1 to the Golgi is sensitive to nocodazole and brefeldin A. Co-expression of a PRA1 export mutant or knockdown of PRA1 led to redistribution of LMP1 and its associated signaling molecules from the Golgi to the ER and subsequent impairment of LMP1-induced NF-kappaB activation. Importantly, the PRA1 knockdown-associated impairment of LMP1 signaling could be restored by reconstitution of the wild-type but not the mutant PRA1. Moreover, neither CD40- and TNFR1-mediated NF-kappaB signaling nor the functional integrity of the Golgi apparatus was affected by PRA1 knockdown, indicating that PRA1 selectively mediates the trafficking and signaling of LMP1. Finally, targeting of the LMP1 C-terminus to the Golgi by fusion with a palmitoylation sequence derived from GAP-43 was insufficient to trigger LMP1-induced NF-kappaB activation unless the LMP1 derivative was able to interact with PPA1, implicating that PRA1 is not merely an interacting-transporter for LMP1, but is crucial for LMP1-mediated signaling. Collectively, these novel findings provide important new insights into LMP1, and identify an unexpected new role for PRA1 in cellular signaling.
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