Signaling pathways are tightly regulated by a dynamic interplay between phosphorylation and protein-protein interactions. Although both events are intrinsically linked, only few technologies allow their simultaneous study.
Using the ERK pathway as model, we first examined the respective impact of MEK1 and ERK2 activation loop phosphorylation on their interaction, by luminescent oxygen channelling assays. MEK1 phosphorylation rather than that of ERK2 has the most significant impact on binding. We then harnessed the channelling assays by combining two chemiluminescent beads bearing distinct dyes to simultaneously monitor phosphorylation and protein-protein interaction in a single well. This allowed direct observation of ERK2 dissociation from MEK1 upon phosphorylation. To further validate this approach, c-Raf-MEK1 and ERK2-Elk-1 were tested with similar outcomes. Dephosphorylation-interaction assays were performed on ERK2 and P38α with three MKPs. The characteristic catalytic and binding patterns generated by MKP-2, MKP-6 and MKP-7 allowed to discriminate mechanisms of action as well as substrate selectivity.
Next, we tested a panel of MAP2Ks against classic MAP kinases. As expected, cognate pairs displayed phosphorylation and interaction. Interestingly, MKK6 binds ERK1 and ERK2. Although this interaction can be modulated by ATP, ERK1/2 are not phosphorylated by MKK6. The MEK1-ERK2 interaction can also be modulated by ATP in a phosphorylation-independent manner, and this requires the integrity of MEK1 catalytic domain. Based on the observation that nucleotides can perturb MEK1-ERK2 binding, phosphorylation-interaction experiments were performed in the presence of small molecule inhibitors. The patterns obtained can be used to discriminate between an ATP-competitor and an allosteric MEK modulator.
Thus, combining single-well measurements of catalytic activity and substrate binding provides biochemical insight and can be used to determine mechanism of action in a drug discovery endeavour.