MAPK-interacting protein kinases 1 and 2 (MNK1 and MNK2) function downstream of p38 and ERK MAPK, but there are large gaps in our knowledge of how MNKs are regulated and function. As proteins activated in the HER2/Ras/Raf/ERK pathway, the MNKs are of potential interest in HER2-overexpressing cancers. We utilized a panel of breast cell lines (HCC1419, AU565, SKBR3, MCF7, and MCF10A), three of which overexpress HER2, to characterize the amounts and activation status of MNKs and other pathway enzymes (ERKs and RSKs) in these cells. We generated a phosphospecific antibody to Thr(P)-214 in the T-loop of MNKs and found that phosphorylations of both Thr-209 and Thr-214 in human MNK1 are required for activation. Increased phosphorylation and activity of the MNKs correlate with HER2 overexpression, and inhibition of the MNKs reduces colony formation in soft agar. Our work identifies the MNKs as potential therapeutic targets for breast cancer treatments.The functional role of MAP 4 kinase-interacting kinase 1 (MNK1) and MNK2 in signal transduction is one of the major unsolved problems in signal transduction (1), and only recently has a MNK1 function been shown in translation of cytokines (2). Likewise, there are still large gaps in our understanding of how MNKs are regulated by MAP kinases. Human MNK1 was cloned as a novel substrate for ERK1, and the phosphorylation site identified was Thr-334 in the C terminus (3).MNK1 and MNK2 contain a canonical Thr-Pro motif for MAPKAP kinases in the T-loop, nine residues N-terminal to the conserved APE motif. Phosphorylation of the canonical Thr-Pro motif is required for activation of the RSK C-terminal domain (4), the MSK1 C-terminal domain (5), and MAPKAP kinase 2 (6). For these MAPKAP kinases, the canonical phosphorylation is the only phosphorylation required in the T-loop. However, MNK1 and MNK2 have atypical T-loops as the DFG motif is replaced by DFD, and in the only structures available (MNK2 (7)), the MNK T-loop has out-in conformations requiring a conformational switch to coordinate with Mg 2ϩ /ATP. In the T-loop of MNKs, there is a second Thr-Pro motif, which is conserved even in Drosophila melanogaster Mnk (7). The double alanine mutant of huMNK1 is inactive (T209A/T214A), and the T-loop is phosphorylated at both sites, as assessed by Thr to Ser mutations, peptide mapping, and phosphoamino acid analyses of resolved peptides (8). The available phosphospecific antibody (Cell Signaling) was made to a proprietary phosphopeptide containing both Thr-209/Thr-214 phospho-sites. The role of the canonical Thr-214 site, in particular, has not been deconvoluted and deserves more study. We investigated regulation of MNK phosphorylation with a phosphospecific antibody we generated for the canonical Thr-214 -Pro as well as the available phospho-Mnk1 (Thr-197/202) antibody (Cell Signaling).Human epidermal growth factor receptor 2 (HER2) (also known as ERBb2/Neu) is frequently overexpressed and/or mutated in aggressive breast cancers (9). HER2 is the molecular target of trastuzumab (Hercep...

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