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

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites—short linear motifs found in MAPK substrates, regulators, and scaffolds—can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity. High throughput yeast two hybrid Illumina sequencing to determine the interactions of ERK2 and p38α with a library of the ERK2 substrate ELK1 with a variable docking site sequence. In the library the native MAPK docking sequence in ELK1 is replaced by short linear motifs found in human proteins. Library representation is evaluated after two cycles of competitive selection. Extracted DNA from each aliquot and the D-site coding sequence was PCR-amplified with barcoding primers. Samples were pooled and sequenced on an Illumina HiSeq 4000 instrument with 100-bp paired end reads. Screens with ERK2 and p38α were performed three times independently.