MNK1 Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
The MAP kinase interacting serine/threonine kinase 1 (MKNK1; MNK1) activates translation of proliferation associated proteins by phosphorylation of elongation initiation factor 4E (eIF4E) at serine 209. MNK1 itself is phosphorylated and activated by the p38 kinases and kinases in the Erk pathway downstream of many receptor tyrosine kinases.
The human T cell lymphoma cell line Karpas-299 expresses a constitutively active fusion protein of the receptor tyrosine kinase ALK (NPM-ALK) which activates the ERK pathway and subsequently MNK1, resulting in phosphorylation of eIF4E at Ser209. Inhibition of MNK1 with the cognate MNK1 inhibitor CGP-57380 results in a decrease of phospho-levels of MNK1 substrate eIF4E (see Fig. 1). The cellular eIF4E phosphorylation assay is suited for the characterization of inhibitors of MNK1. In the assay, levels of phospho-Ser209 of eIF4E are quantified by Sandwich-ELISA technique.
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
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Reference compound IC50 for MNK1
The known MNK1 Inhibitor CGP-57380 (4-Amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine) acted as inhibitor of phosphorylation of eIF4E at Ser209 in Karpas-299 cells and generated highly reproducible IC50 values in the cellular MNK1 activity assay. The graph shows a representative result.