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. 2002 May;76(9):4567–4579. doi: 10.1128/JVI.76.9.4567-4579.2002

FIG. 3.

FIG. 3.

Tpl-2 modulates LMP1-induced NF-κB activation. (A) Wild-type Tpl-2 induces levels of NF-κB transactivation similar to that of NIK. NIH 3T3 cells were transfected with a β-galactosidase-expressing plasmid and an NF-κB reporter in the presence of increasing amounts of NIK or Tpl-2 and 36 h later were analyzed for luciferase and β-galactosidase activities. Relative NF-κB activation (ratio of luciferase versus β-galactosidase activities; RLV) from two independent experiments is shown. Asterisks represent individual mean values of duplicate determinations from these experiments. (B) Kinase-inactive Tpl-2 inhibits LMP1-induced NF-κB binding activity in 293 cells, as determined by EMSAs (upper panel). The NF-κB complex shown contains p50-p65 heterodimers (data not shown). Nuclear extracts were also analyzed for Sp1 binding activity as a control (lower panel). (C) Kinase-inactive Tpl-2 inhibits LMP1-induced NF-κB transactivation. NIH 3T3 or HEK 293 cells were cotransfected with a galactosidase-expressing plasmid and an NF-κB reporter and 1 μg of pSG5-LMP1, in the presence of equivalent amounts of Tpl-2[K167M]. Relative NF-κB activation (ratio of luciferase versus β-galactosidase activities) (± standard deviation [SD]) from three independent experiments is shown. (D) Kinase-inactive Tpl-2 inhibits LMP1-, CTAR1 [LMP1Δ(332-386)]-, and CTAR2 (LMP1AxAxA)-induced NF-κB transactivation to similar extents. HEK 293 cells were transfected with 1 μg of pSG5-based vectors in the presence of reporter constructs and increasing amounts of Tpl-2[K167M]. To allow comparison of the inhibitory effect of kinase-inactive Tpl-2, RLVs have been normalized to 100 for each of the wild-type, CTAR1, and CTAR2 effects. In these experiments, the mean NF-κB activation values from the above LMP1 molecules were 60 (±5.4), 48.4 (±6.2), and 13 (±4). (E) Kinase-inactive GCKR does not affect LMP1-induced NF-κB transactivation. HEK 293 cells were transfected with reporter constructs and 1 μg of pSG5-LMP1, in the presence or absence of increasing amounts (0.25 or 0.5 μg) of dominant negative GCKR (GCKRdn). RLV was assessed as described for panel C. (F) Kinase-inactive Tpl-2 does not affect Cdc42-mediated NF-κB induction. HEK 293 cells were transfected with reporter constructs and 5 μg of pcDNA3-Cdc42, in the presence or absence of increasing amounts (0.25 or 0.5 μg) of dominant negative Tpl-2. RLV was assessed as described for panel C.