CKLiK activates ERK1 MAP kinase but not PKB.

(A) HA_PKB or HA_ERK1 (2 μg) was transfected in COS cells with or without CaMKKα (8 μg) or CKLiK-296 (8 μg) as indicated. Twenty hours before harvesting cells were serum starved. Unstimulated and 20% FCS-treated cells were used as controls. HA_PKB or HA_ERK were immune precipitated and kinase activity was measured using histone 2B (H2B) or myelin basic protein (MBP) as a substrates. Data represent 1 of at least 4 independent experiments. (B) HA_ERK1 or HA_PKB (2 μg) was cotransfected with ER_CKLiK-296 (8 μg) in COS cells. Before harvesting, cells were stimulated with 4-OHT for 2 or 10 minutes. Immunoprecipitations and kinase assays were performed as in panel A using H2B or MBP as a substrate. Equal expression of HA_PKB and HA_ERK were analyzed by Western blotting of whole cell lysates with 12CA5 antibody. (C) PD098059 inhibits the CKLiK-induced ERK activity. COS cells were transfected with HA_ERK1 (2 μg) and ER_CKLiK-296 (8 μg). Cells were treated with or without the MEK inhibitor PD098059 (50 μmol/L) for 15 minutes before stimulation with 4-OHT for indicated time periods. Cells were lysed and immunocomplex kinase assays were performed as previously described. Equal expression of HA_ERK1 was analyzed by Western blotting.