Figure 8.

The amino-terminal extension of JNKK1 is required for transducing upstream stimuli to JNK in intact cells. (A) The amino-terminal extension is required for inhibition of JNK activation by dominant-negative JNKK1. HeLa cells were cotransfected with HA–JNK2 and ΔMEKK1 expression vectors, along with either empty expression vector (−) or expression vectors for FL(1–399) GST–JNKK1(AA) or amino-terminally truncated (78–399 and 89–399) GST–JNKK1(AA). After 48 hr, JNK activity was determined by immunecomplex kinase assays (KA) using anti-HA antibody and GST–c-Jun(1–79) as a substrate (top panel). The content of GST–JNKK1(AA) proteins and HA–JNK2 was determined by immunoblotting (IB; bottom two panels). (B) The amino-terminal extension of JNKK1 is required for response to stimuli. HeLa cells were transfected with expression vectors for either FL(1-399) or amino-terminally truncated (78–399) GST–JNKK1. After 48 hr, the cells were incubated with either 0.4 m sorbitol (osmotic shock) or 20 ng/ml of TNF for 20 min. Cell lysates were prepared, the GST–JNKK1 proteins were precipitated with GSH–Sepharose, and their kinase activities were determined. (C) The amino-terminal extension of JNKK1 is required for inhibition of JNK activation in response to physiological stimuli. HeLa cells were cotransfected with HA–JNK2 and either an empty vector (−) or expression vectors for catalytically inactive ΔMEKK1 [MEKK1(KM)] and either FL(1–399) or amino-terminally truncated (78–399) GST–JNKK1(AA). After 40 hr, the cells were exposed to anisomycin (15 ng/ml), UV-C (20 J · m⁻²), TNF (5 ng/ml), or EGF (20 ng/ml). Lysates were prepared after 20 min and HA–JNK2 kinase activity was determined as described above. (D) A sequential-interaction model for organization of the MEKK1–JNKK1–JNK(p38) MAPK module. MEKK1, either active or inactive, interacts with inactive JNKK1 to form a MEKK1:JNKK1 complex, whose formation depends on the amino-terminal extension of JNKK1. Activated MEKK1 phosphorylates and activates JNKK1, resulting in dissociation of the MEKK1:JNKK1 complex. Activated JNKK1 then interacts with JNK (or p38) through its amino-terminal extension. JNK (or p38) activation is followed by dissociation of the JNKK1:JNK complex and activated JNK is freed to bind its targets and phosphorylate them.