Fig 4. NleL targets other human JNK family proteins (JNK2 and JNK3).

(A) A schematic view of the sequence homology between human JNK family proteins. The level of sequence homology between JNK1 and JNK2 or JNK3 (90% and 96%, respectively) is indicated. (B) NleL interacted with JNK2. Flag-tagged JNK2 and His₆-tagged NleL (His-NleL) or C753A mutant (His-NleL-CA) were ectopically expressed in cells. Cells were lysed and subjected to Co-IP with anti-Flag beads, followed by IB analysis with indicated antibodies. (C) NleL interacted with endogenous JNKs or ectopically expressed JNK3. GST-tagged NleL, its C753A mutant or GST only was individually subjected to the GST pull-down assay with cell lysates of HEK293T cells transfected with (right) or without (left) Flag-tagged JNK3. The bound proteins were immunoblotted with anti-JNK antibody. (D) NleL promotes ubiquitylation of JNK2 (left) or JNK3 (right) in vivo. (E) NleL blocked TNFα-induced JNK1, JNK2 or JNK3 phosphorylation. HEK293T cells expressing Flag-tagged JNK1, JNK2 or JNK3 and His₆-tagged wild-type NleL or its C753A mutant were subjected to TNFα treatment (10 ng/ml) at indicated times. Phosphorylation status of JNKs was determined by IB analyses with anti-p-JNK. (F and G) NleL-associated JNK2/3 ubiquitylation conversely correlates with the phosphorylation of JNK2/3. Phosphorylation and ubiquitylation status of JNK2 (F) or JNK3 (G) was assayed with Flag-JNKs enriched from cells expressing wild-type NleL or the C753A mutant, followed by immunoblotting analyses with indicated antibodies. The in vitro kinase assay was performed with Flag-tagged JNK2 (F). All the blots are representative of at least three independent experiments.