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Uncovering Novel MAPK Interactors: A Novel Yeast-based Genetic Screening Platform

In a recent study published in Science, researchers have developed a yeast-based genetic screening pipeline for evaluating large numbers of mitogen-activated protein kinase (MAPK) docking sequences in parallel.  This approach was used to characterise MAPK docking sequences, defining critical features that allow molecules to bind to MAPKs.

The role of protein kinases

Protein kinases selectively modify other proteins by covalently attaching phosphates to them at specific effector sites – a process known as phosphorylation. Protein kinases can achieve specificity in a number of ways, such as by matching the shape of the enzyme’s active site (called the catalytic cleft) to the residues surrounding the site of phosphorylation. However, some protein kinases can only recognize very simple patterns on the proteins they modify – and this may not be specific enough to only target one type of protein.

The mitogen-activated protein kinases (MAPK) family are a group of serine-threonine kinases that are conserved widely in eukaryotes and play a key role in cell signalling. MAPKs are activated in response to diverse cellular stimuli, and the different MAPK subfamilies phosphorylate unique effector sites to elicit distinct cellular responses. One thing all MAPKs have in common is that they target a Ser/Thr-Pro consensus sequence. The specificity of MAPKs is partly driven by docking interactions (non-catalytic interactions), in which regions of the kinase outside of the catalytic cleft recruit substrates through binding sites away from sites of phosphorylation.

A region called the D-recruitment site (DRS) is a specific region on MAPKs that binds to other molecules, such as substrates, scaffold proteins, MAPK kinases (MKKs) and MAPK phosphatases (MKPs). This region recognizes short linear motifs (SLiMs) called D-sites, which are found in the unstructured regions of molecules that interact with MAPKs. The binding of D-sites to MAPKs are transient. This allows for dynamic remodelling of signalling networks in response to stimuli. The DRS can also alter the conformation of the catalytic cleft of MAPKs, which allows MKKs to activate MAPKs, and MKPs to inactivate them.

Discovering new MAPK interactors

In this study, researchers developed a new yeast-based genetically encoded library screening platform to identify new MAPK interacting D-sites. This is possible because the cascades involving MAPKs are conserved between humans and yeast (and amongst almost all eukaryotes). A library consisting of ~12,000 sequences from the human proteome was screened, revealing a large number of MAPK-selective interactors, including many that do not conform to previously defined docking motifs. By reconstituting signalling pathways in yeast, this allowed researchers to tune pathway inhibition through competitive interactions between kinases in the MAPK cascade or with downstream effectors.

Unlike previous methods, this approach specifically focuses on MAPKs and docking interactions. Moreover, it allowed for the identification of interactions that occur within a eukaryotic cell, providing a more physiologically relevant context for the interactions. Additionally, this approach is high-throughput, enabling the extraction of binding motifs and the discovery of interaction partners that might escape detection in other experiments due to low abundance or restricted patterns of expression. However, there are some limitations – this approach may fail to identify lower affinity interactions and known interactors that have additional sequences that flank the D-site. Overall, this novel approach could be a powerful new tool for the discovery of MAPK interactors, and useful in understanding the regulation of MAPK signalling pathways.

More on these topics

cell signalling / Genomics / Yeast