PKCζ directly phosphorylates ERK5 in vitro. (A) To determine direct PKCζ-induced ERK5 phosphorylation, we performed an in vitro kinase assay with 4 different GST-ERK5 fragments as substrate. In vitro kinase assay shows 32P incorporation into 2 GST-ERK5 fragments (amino acids 100 ∼ 200 and 400 ∼ 600) but not into the others (amino acids 200 ∼ 400 and 600 ∼ 816). Ponceau staining shows the position of the proteins after separation by SDS-PAGE and near equal expression. (B-C) Characterization of PKCζ phosphorylation sites. In vitro kinase assay was performed with ERK5-WT fragment (aa1-200 or aa400-600) and ERK5 fragment with S31A (B) or S486A (C) mutations in the presence of recombinant PKCζ. Mutation of the serine 486 to alanine in GST-ERK5 200-600 ablated PKCζ-mediated ERK5 phosphorylation. Data presented are from a representative experiment of at least 3 independent experiments.
