Figure 5.

Phosphorylation of JNKK1 disrupts its binding to ΔMEKK1. GST-fusion proteins containing wild-type JNKK1, an ATP-binding mutant, JNKK1 (KR), or a mutant lacking the activating phosphoacceptor sites, JNKK1(AA), were transiently expressed in Cos-1 cells. Lysates containing these proteins were mixed with ΔMEKK1-containing lysates and incubated for 20 min at 30°C in kinase buffer without ATP or with 1 mm ATP or AMP–PNP as indicated. Next, the GST fusion proteins were precipitated with GSH–Sepharose. After washing six to eight times, the precipitates were resolved by SDS-PAGE and immunoblotted with anti-MEKK1 (top) and anti-GST (bottom).