Mitogen activated protein kinases (MAPKs) have a docking groove that interacts with linear motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of fifteen linear motifs from diverse MAPK partners to bind to c-Jun N-terminal kinase 1 (JNK1), p38α and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus could differentiate between the topographically similar docking grooves of ERK and p38. We also solved the crystal structures for four MAPK-docking peptide complexes that represented JNK-specific, ERK-specific or ERK-and p38-selective binding modes. These structures revealed that the regions located in between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a non-discriminatory fashion, specificity was determined mainly by the conformation of the intervening region between the anchor points. These insights enabled us to successfully design peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of docking motif regions located between anchor points. We present a coherent structural model underlying MAPK docking specificity that reveals how short linear motifs

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.protein kinase | signal transduction | structural biology | ERK2 | RSK1

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