oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain

TRAF6 mediates JNK and p38 activation by IL-1. (A) HEK293 cells were cotransfected with HA-JNK1 (0.5 μg/plate) along with either an empty expression vector (Vec), IRAK or
IRAK(Δ218–507) (100 ng/plate each), or TRAF6 or TRAF6(289–522) (1 μg/plate each) expression vectors. Total DNA was kept constant
(1.5 μg/plate) using empty expression vector. After 24 hr the transfected cells were treated with IL-1 (10 ng/ml) for 30 min
or left untreated. Cells were collected, lysed, and HA–JNK1 activity was determined by immunocomplex kinase assay with GST-cJun(1–79)
as a substrate. Fold-increase in HA–JNK1 activity above the basal level in cells cotransfected with empty expression vector
was determined by PhosphorImaging and normalized to the level of HA–JNK1 expression, determined by immunoblotting. (B) HEK293 cells were transfected as described above except that an HA–p38α vector (0.5 μg/plate) was used instead of the HA–JNK1
vector. HA–p38α activity was determined, as above, by immunocomplex kinase assay with myelin basic protein (MBP) as a substrate.
(C) HEK293 cells were cotransfected with HA–JNK1 (0.5 μg/plate), IRAK or IRAK(Δ218–507) (100 ng/plate each), TRAF6 or TRAF6(289–522)
(1 μg/plate each) expression vectors as indicated. After 24 hr some cultures were treated with IL-1 for 30 min and the rest
left untreated. HA–JNK1 activity and expression were determined as described above. Expression of Flag–TRAF6, Flag–TRAF6(289–522),
or IRAK was determined by immunoblotting.

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