FIG. 3.

cAMP differentially regulates MEK5 and MEK1. (A) C2C12 myoblasts grown to confluence and cultured in 0.5% FBS for 24 h were treated with DMSO or 10 μM forskolin and 50 μM IBMX for 15 min and then with 1 ng/ml EGF for 5 min. MEK5 was immunoprecipitated from cells, and activity was determined using a coupled in vitro kinase assay with GST-ERK5 (1-451) and GST-MEF2C (204-321). Changes in MEK5 activity are reflected in changes in GST-ERK5 (1-451) activity towards GST-MEF2C (204-321). (B) HeLa cells grown to confluence and cultured in 0.5% FBS for 24 h were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 2 or 5 min with 1 ng/ml EGF. MEK1 activity was determined with immune complex kinase assays using GST-ERK2KR as the substrate. Results are representative of at least two independent experiments. (C) HeLa cells as described for panel B were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 5 min with 100 nM phorbol ester (phorbol myristate acetate). MEK1 activity was determined with immune complex kinase assays using GST-ERK2KR as the substrate. Results are representative of at least two independent experiments.