LMP1 activates NF-kappa B via degradation of I kappa B alpha in nasopharyngeal carcinoma cells

Objective To elucidate the mechanisms by which Epstein-Barr virus-encoded l atent membrane protein 1 activates NF-kappaB in nasopharyngeal carcinoma ce lls. Methods A tetracycline-regulated LMP1-expressing nasopharyngeal carcinoma c ell line, Tet-on-LMP1-HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, I kappaB alpha and I kappaB beta, was analyzed by Western blotting. The subcellular localization of NF-kappaB (p65) was detected by indirect immunofluorescence assay. The NF-kappaB tra nsactivity was studied by transient transfection and reporter gene assay. Results I kappaB alpha was phosphorylated and degraded after the inducible expression of LMP1, although the total protein levels remained stable. The steady-state level of total I kappaB beta protein may have resulted from th e initiation of an autoregulation loop after the activation of NF-kappaB. N o change in the I kappaB beta level was detected. NF-kappaB (p65) was trans located from the cytoplasm to the nucleus following degradation of I kappaB alpha. After the introduction of the dominant-negative mutant Of I kappaB alpha (Del 71) into Tet-on-LMP1-HNE2 cells, both nuclear translocation and transactivation of NF-kappaB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, L MP1 activated NF-kappaB via phosphorylation and degradation of I kappaB alp ha, but not I kappaB beta. The dominant-negative mutant of I kappaB alpha ( Del 71) could completely inhibit both the nuclear translocation and transac tivation of NF-kappaB induced by LMP1.