MNK1, a new MAP kinase‐activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates

We have developed a novel expression screening method for identifying protein kinase substrates. In this method, a λ phage cDNA expression library is screened by in situ, solid‐phase phosphorylation using purified protein kinase and [γ‐32P]ATP. Screening a HeLa cDNA library with ERK1 MAP kinase yielded cDNAs of previously characterized ERK substrates, c‐Myc and p90RSK, demonstrating the utility of this method for identifying physiological protein kinase substrates. A novel clone isolated in this screen, designated MNK1, encodes a protein‐serine/threonine kinase, which is most similar to MAP kinase‐activated protein kinase 2 (MAPKAP‐K2), 3pK/MAPKAP‐K3 and p90RSK. Bacterially expressed MNK1 was phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 was activated upon stimulation of HeLa cells with 12‐O‐tetradecanoylphorbol‐13‐acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor‐α, interleukin‐1β or osmotic shock, and the activation by these stimuli was differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways.

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