Mp119:arcand:ATP MAP Kinase Multiplex

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A Homogenous Multiplexing Assay Studying the Phosphorylation-Interaction Interplay Between MAP2Ks and MAP kinases

Mathieu Arcand, Roger Bossé, Philippe Roby and Sophie Dahan

Bio-discovery, PerkinElmer, 1744 William Suite 600, Montreal, Quebec, Canada H3J 1R4

MAP kinase pathways are essential to numerous biological processes that include growth and inflammation. These pathways are tightly regulated by interplay between phosphorylation events, protein-protein interactions and subcellular localization. For example, in the ERK MAP kinase pathway, the upstream MAP2K, MEK1, binds to MAP kinase ERK2. This interaction occurs in the cytoplasm with both proteins unphosphorylated. Upon sequential phosphorylation, the kinases become active; this in turn causes their dissociation and subsequent ERK2 nuclear localization.

By multiplexing AlphaScreen® and AlphaLISA® beads, we have recently observed in a single well the direct dissociation of ERK2 from MEK1 upon phosphorylation. Furthermore, we have used this experimental approach to discriminate an allosteric inhibitor from an ATP competitor.

Here, we use these tools to further study the protein-protein interactions between MEK1 and ERK2 with respect to phosphorylation state-dependent and -independent effects.

We first determined that the dissociation between the two proteins is solely dependent on their activation state and does not involve a phosphorylation feedback loop. Second, by co-titration experiments on full-length recombinant proteins, we confirmed that phosphorylation of MEK1 and/or ERK2 reduces their binding. However, this interaction is more sensitive to the phosphorylation of MEK1 than that of ERK2. Interestingly, the binding between unphosphorylated MEK1 and ERK2 is impaired by increasing ATP concentrations. This interaction modulation requires the intact catalytic domain of MEK1 but not that of ERK2. Other phospho-nucleotides such as ADP, ATP-γ-S, and UTP can also modulate MEK1-ERK2 binding, whereas AMP and GTP have no effect. Finally, ATP can modulate the interaction between ERK2 and its non-cognate MAP2K MKK6 also in a phosphorylation-independent manner.

The above results suggest that nucleotides can influence the conformation of MAP2Ks. These changes affect MAP kinase binding and our Alpha technologies platform provided a sensitive approach to detect this subtle modulation in protein-protein interactions. More so, multiplexing phosphorylation and interaction events allows screening for small molecules that modulate catalytic activity and enzyme-substrate interactions, either individually or together.

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