FIG. 5.

cAMP inhibition of MEKK2 requires PKA. (A) HeLa cells grown to confluence and cultured in 0.5% FBS for 18 h were treated with 5 μM H-89 for 1 h, then with DMSO or 10 μM forskolin and 50 μM IBMX for 15 min, and then for 5 min with 100 ng/ml EGF. (Top) MEKK2 immune complex kinase assay using His-MEK6KM as substrate. MEKK2 autophosphorylation and MEKK2 activity towards MEK6KM are seen. (Middle) MEKK2 immunoblot of MEKK2 immune complex kinase assay. (Bottom) ERK5 activity was monitored as described for Fig. 1. Results are representative of at least three independent experiments. (B) HeLa cells were transfected with no siRNA, 10 nM siRNA directed toward MEKK2, or 40 nM siRNA directed toward ERK1/2. After 48 h, cells were transferred to 0.5% FBS for 18 h and then left untreated or stimulated for 15 min with 1 ng/ml EGF. (Top) ERK5 activity monitored as described for Fig. 1. (Middle two panels) MEKK2 (second from top) and ERK1/2 (third from top) immunoblots of cell lysates. (Bottom) ERK1/2 activity monitored as described for Fig. 1. Results shown in panels A and B are representative of at least three independent experiments.