Structural Mechanisms of Allostery and Autoinhibition in JNK Family Kinases

c-Jun N-terminal (JNK) family kinases have a common peptide-docking site used by upstream activating kinases, substrates, scaffold proteins, and phosphatases, where the ensemble of bound proteins determines signaling output. Although there are many JNK structures, little is known about mechanisms of allosteric regulation between the catalytic and peptide-binding sites, and the activation loop, whose phosphorylation is required for catalytic activity. Here, we compare three structures of unliganded JNK3 bound to different peptides. These were compared as a class to structures that differ in binding of peptide, small molecule ligand, or conformation of the kinase activation loop. Peptide binding induced an inhibitory interlobe conformer that was reversed by alterations in the activation loop. Structure class analysis revealed the subtle structural mechanisms for allosteric signaling between the peptide-binding site and activation loop. Biochemical data from isothermal calorimetry, fluorescence energy transfer, and enzyme inhibition demonstrated affinity differences among the three peptides that were consistent with structural observations. ► Peptide binding induces two distinct autoinhibitory mechanisms in the inactive JNKs ► Activation loop conformer can reverse peptide-induced autoinhibition ► Bidirectional signals are visualized between peptide and activation loop ► Structural class analysis reveals subtle structural features The c-jun N-terminal-kinase (JNK) family phosphorylates many substrates and interacts with many scaffold proteins. Laughlin et al. compare crystal structures of JNK3 with three different peptides derived from either scaffold proteins or substrates and reveal an allosteric signaling mechanism of autoinhibition.