Fig. 1: Overview of Human MKK–MAPK interactions and screening approach for MKK1-ERK2.

a A schematic overview of cognate MKK interactions with MAPKs and their downstream effectors. The D-domain is visualised as the multi-colour section of the unstructured N-terminal region of MKKs. b Phosphorylation of ERK1/2 by MKK1/2 is facilitated by MKK’s D-domain binding the D-domain recruitment site (DRS) of ERK2. Shown here is the D-domain sequence of MKK2, the residues highlighted in red bind an acidic patch of residues in the ERK2 DRS, while the large hydrophobic residues highlighted in aqua bind hydrophobic grooves within the ERK2 DRS. Spacer residues are shown in pink (PDB: 4H3Q)¹⁵. c (1) Paramagnetic beads (green circle) are functionalised with subGFP (blue wavy line with GFP) and ^caMKK1 genes (pink wavy lines). (2) Beads are emulsified by mixing an aqueous solution containing in vitro expression components (IVTT) and purified ERK2 protein with oil and surfactant. (3) Expression of ^caMKK1 in the emulsion droplets starts the cascade by phosphorylation of ERK2. Phosphorylated ERK2 is now able to phosphorylate serine within the substrate sequence of the subGFP construct. (4) After de-emulsification, beads are exposed to chymotrypsin protease (denoted as scissors) in bulk. (5) Beads with phosphorylated serine will be less susceptible to digestion by chymotrypsin, leaving subGFP attached to the bead. The bead, therefore, encompasses both the genotype and phenotype information, as an encoded active ^caMKK1 mutant will retain more subGFP on a bead than a less-active kinase mutant. (6) Subsequent flow cytometric sorting of the beads based on subGFP fluorescence into activity-set gates followed by (7) deep next-generation sequencing (NGS) allows for correlation of the cascade activity to the encoded MKK1 gene.