FIG. 1.
A lysineless mutant of ERK3 is unstable. (A) Schematic representation of ERK3Δ constructs showing the position of lysines mutated in this study. (B) HEK 293 cells were transfected with the indicated constructs. After 48 h, cell lysates were analyzed by immunoblotting with anti-HA antibody. Two different exposures of the gel are shown (upper two panels). The same constructs were translated in vitro in the presence of 35S-labeled amino acids and were analyzed by fluorography (lower panel). (C) HEK 293 cells transfected with the different ERK3 constructs were treated with cycloheximide (100 μg/ml) for the indicated times. Ectopically expressed ERK3 was detected by HA immunoblotting. (D) HEK 293 cells were transfected with Myc6-tagged ERK3Δ, ERK3Δ kinase dead (KD), ERK3Δ Ser189Ala (S189A), ERK3Δ-0K, or ERK3Δ1-296. The cells were metabolically labeled with 32P, and the transfected ERK3 proteins were immunoprecipitated with anti-Myc antibody. Phosphorylation was revealed by autoradiography (upper panel). Aliquots of cell lysates were analyzed by immunoblotting to monitor expression of the ectopic proteins (lower panel). exp., exposure; IB, immunoblot; IP, immunoprecipitation.