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Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

ABSTRACT: MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.

SUBMITTER: Bardwell AJ

PROVIDER: S-EPMC4646321 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

Bardwell A Jane AJ Bardwell Lee L

The Journal of biological chemistry 20150914 44

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docki ...[more]

PMID: 26370088

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Project description:UnlabelledTwo-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins.ImportanceUsing in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems.

Project description:Mitogen-activated protein kinases (MAPK) are important regulatory units in cells and they take part in the regulation of many cellular functions such as cell division, differentiation or apoptosis. All MAPKs have a shallow docking groove that interacts with linear binding motifs of their substrate proteins and their regulatory proteins such as kinases, phosphatases, scaffolds. Inhibition of these protein-protein interactions may reduce or abolish the activity of the targeted kinase. Based on the wide range of their biological activity, this kind of inhibition can be useful in the treatment of many disorders like tumors, inflammation or undesired cell apoptosis. In this study a linear binding motif from the RHDF1 protein-a 15 amino acids long peptide-was selected for optimization to increase its cellular uptake but retaining its low micromolar binding affinity. First, we synthesized an octaarginine conjugate that showed efficient cellular uptake. Next, we set out to reduce the size of this construct. We were able to decrease the length of the original peptide, and to increase its cellular uptake with specific chemical modifications. These new constructs bound better to ERK2 and p38 kinases than the original peptide and they showed markedly increased cellular uptake. The new octaarginine conjugate and one of the minimized bicyclic derivatives could inhibit the phosphorylation of intracellular ERK or p38. However, the modulation of MAPK phosphorylation levels by these cell-penetrating peptides were complex, despite that in biochemical assays they all inhibited MAPK-substrate binding as well as phosphorylation. The optimized peptides depending on the applied concentration caused an expected decrease, but also some unexpected increase in MAPK phosphorylation patterns in the cell. This possibly reflects the complexity of MAPK docking groove mediated protein-protein interactions including bone fide MAPK clients such activator kinases, deactivating phosphatases or regulatory scaffolds. Thus, our findings with optimized cell-penetrating "inhibitory" peptides highlight the opportunities but also the pitfalls of docking peptide based MAPK activity regulation and call for a better quantitative understanding of MAPK mediated protein-protein interactions in cells.

Dataset Information

Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

Publications

Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

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