Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases

Abstract

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to a wide array of stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling specific inputs to appropriate cellular responses. One way that MAPKs achieve specificity is through transient interactions with docking sites: short linear motifs found in MAPK substrates, regulators, and scaffolds. Docking sites bind to a conserved groove located in the catalytic domain of all MAPKs including the ERK and p38 subfamilies, but how specificity is achieved remains unresolved. To understand the basis of docking selectivity for these two subfamilies, we screened a library of thousands of human proteome-derived sequences for docking to ERK2 and p38α. We discovered a large number of sequences that bound specifically to only one MAPK or promiscuously to both, and that selective and non-selective interactors conformed to distinct sequence motifs. In particular, selective binding to p38α correlated with higher net charge in the docking site, and this phenomenon was driven by enrichment for Lys residues. A pair of acidic residues unique to the docking groove of p38α mediated selectivity for Lys-rich basic motifs. Finally, we validated a set of full-length proteins harboring docking sites selected as hits in our screens to be authentic MAPK interactors and identified ChREBP and TACC1 as cellular MAPK substrates. This study identifies distinguishing features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

Residues Neighboring an SH3-Binding Motif Participate in Determining Affinity and SpecificityIn Vivo

“…More recently, studies have begun to use techniques such as deep mutational scanning (DMS) to study the impact of large libraries of mutations on protein stability and function in vivo (Fowler and Fields 2014). Methods such as protein complementation assays (Tarassov et al 2008;Michnick et al 2016) have also successfully been used to measure the in vivo binding strength of libraries of mutants in yeast (Diss and Lehner 2018;Dionne et al 2021;Faure et al 2022;Robles et al 2023;Bendel et al 2023;Dibyachintan et al 2024). For example, a recent study showed the effect on binding of combining mutations in a PDZ domain and its binding motif (Zarin and Lehner 2024).…”