Characterization of MAP kinase docking specificity with yeast genetic screens

Abstract

Mitogen‐activated protein kinase (MAPK) pathways play pivotal roles in making vital cellular decisions. In order to carry out their unique functions, each MAPK recognizes its own repertoire of substrates. Aside from a rather degenerate phosphosite motif (S/T‐P), MAPKs utilize a non‐catalytic site docking interaction to specifically recognize their substrates as well as their regulators. Docking interactions occur between a linear docking site peptide (D‐site) and a conserved docking groove on the MAPK. While a general D‐sites sequence motif has been described, unique features of sequences that specifically target individual MAPKs is not completely understood. Investigations into these interactions is difficult due to the weak and transient nature of the interaction.Here I present screening methods using S.cerevisiae to identify functional docking site sequences that direct to specific MAPKs. I found that co‐expressing a mammalian MAPK and its MKK activator in yeast leads to growth suppression. I have used this phenomenon as the basis of a pooled yeast genetic screen, where cells expressing MKK variants compete in one pooled culture. The change in representation of every single D‐site in the culture over time is monitored by next‐generation sequencing. Variants having more functional D‐sites activate the pathway better, grow slower, and are depleted from the population. I generated and screened a library of ~12,000 candidate D‐sites found in the human proteome against the MAPKs p38α and JNK1.These screens revealed sequence motifs that allow discrimination between the two MAPKs, and also defined a D‐site dependent MAPK interactome which contains many new targets that could broaden our understanding of MAPK biology. Our screens serve as guides to more completely understand weak interactions that play an important factor in the dynamic nature of MAPK signaling networks.Support or Funding InformationThis research was supported by National Institutes of Health grant R01 GM104047.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.