Structure and dynamics of the MKK7–JNK signaling complex

Abstract

Signaling specificity in the mitogen-activated protein kinase (MAPK) pathways is controlled by disordered domains of the MAPK kinases (MKKs) that specifically bind to their cognate MAPKs via linear docking motifs. MKK7 activates the c-Jun N-terminal kinase (JNK) pathway and is the only MKK containing three motifs within its regulatory domain. Here, we characterize the conformational behavior and interaction mechanism of the MKK7 regulatory domain. Using NMR spectroscopy, we develop an atomic resolution ensemble description of MKK7, revealing highly diverse intrinsic conformational propensities of the three docking sites, suggesting that prerecognition sampling of the bound-state conformation is not prerequisite for binding. Although the different sites exhibit similar affinities for JNK1, interaction kinetics differ considerably. Importantly, we determine the crystal structure of JNK1 in complex with the second docking site of MKK7, revealing two different binding modes of the docking motif correlating with observations from NMR exchange spectroscopy. Our results provide unique insight into how signaling specificity is regulated by linear motifs and, in general, into the role of conformational disorder in MAPK signaling.itogen-activated protein kinases (MAPKs) are essential components of eukaryotic signal transduction networks that enable cells to respond appropriately to extracellular stimuli (1). The MAPK signaling pathways feature three sequentially acting protein kinases making up a signaling module: an MKKK (MAPK kinase kinase) that phosphorylates and thereby activates an MKK (MAPK kinase), which then activates the MAPK by phosphorylation. In mammalian organisms, four major MAPK pathways have been identified: extracellular-signal-regulated kinase (ERK1/2), ERK5, p38α/β/γ/δ, and c-Jun N-terminal kinase (JNK1/2/3) (2).Seven human MKKs phosphorylate ERK, p38, and JNK according to the following selectivity scheme: MKK1/2 activate ERK1/2, MKK5 activates ERK5, MKK3/4/6 activate p38, and MKK4/7 activate JNK (3). Signaling specificity is controlled by intrinsically disordered N-terminal regulatory domains of the MKKs (ranging between 40 and 100 aa in length) that selectively bind to their cognate MAPKs (4-8). In the absence of these regulatory domains, phosphorylation of the downstream kinases is extremely inefficient (9, 10). Molecular recognition occurs through so-called docking sites in the regulatory domains composed of two to three basic residues followed by a short spacer of one to six residues and finally a hydrophobic-X-hydrophobic submotif. Crystal structures of ERK, p38, and JNK in complex with docking site peptides show that the basic residues contact the negatively charged common docking groove of the MAPKs, whereas the hydrophobic residues insert into hydrophobic pockets on the surface (Fig. S1A) (7,(11)(12)(13)(14)(15)(16).MKK7 activates the JNK pathway that primarily regulates stress and inflammatory responses. MKK7 is the only human MKK for which three putative docking sites (D1, D2, and D3) are pre...

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase

“…To our knowledge Tau is the first example of ERK2 protein substrate to contain a combination of docking sites. A similar combination of three D-docking sites was described in the regulatory disordered N-terminal region of MKK7 (MAP kinase kinase 7) used for the specific recognition by the JNK MAP kinase (61,62). Another type of a modular system of recognition was previously proposed consisting of a combination of the D-docking site and F-docking site, although in this case it corresponds to two distinct binding grooves of ERK2 (34).…”

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…Thus, the role of JIP1 in the JNK pathway may be mechanistically very different from prototypical signaling scaffolds, such as the Saccharomyces cerevisiae Ste5 protein that increases overall signaling spec-ificity by binding different pathway components simultaneously and so stimulates pathway throughput via allosteric regulatory mechanisms (123). In addition, the mammalian MKK7 shows high specificity for the JNKs (124)(125)(126), and so the mammalian MKK7-JNK pathway would not need to rely on additional scaffold proteins to provide pathway specificity. However, in this context it should be appreciated that scaffold proteins may admit a number of negative and/or positive feedback circuits to set the appropriate level of JNK activity for different subcellular compartments and physiological states.…”

Dynamic Protein Interaction Networks and New Structural Paradigms in Signaling

“…The disordered regulatory domain of MKK7 contains three putative docking sites (D1-3) for the c-Jun N-terminal kinase (JNK), which adopt different conformations, helical, random coil and extended (PPII), in the unbound state. The different intrinsic conformational propensities of the docking sites suggest that there is no need for preformed structural elements sampling the bound state conformations for the JNK binding 105 .…”