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Abstract1 Materials ATP-Competitive versus Allosteric

MEK1 Inhibition

ERK2 – Elk-1 Phosphorylation-

Interaction Assay

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Summary

• Combined use of AlphaScreen® and AlphaLISA® beads allowed

simultaneous monitoring of substrate phosphorylation and

enzyme interaction

• Characteristic phosphorylation-interaction patterns were

generated with three pairs in the ERK MAP kinase pathway

• Raf - MEK1• ERK2 - Elk-1• MEK1 – ERK2

• Dephosphorylation assays allowed to distinguish mechanism of

action and selectivity for three MAP kinase phosphatases

• Simultaneous monitoring of phosphorylation and interaction

events allowed us to discriminate between the mechanisms of

action of two small molecule inhibitors

• ATP-competitor Staurosporine• Allosteric MEK1 inhibitor U0126

• Phosphorylation-interaction patterns can thus be used to

provide significant mechanistic and biological insight

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Simultaneous Monitoring of Phosphorylation Events and Protein-Protein Interactions in the ERK MAP Kinase Pathway

Mathieu Arcand, Philippe Roby, Roger Bossé & Sophie Dahan

The relationship between protein phosphorylation andinteraction events is especially significant in signal transductionpathways. Phosphorylated residues can generate bindinginterfaces, and docking interactions often dictatephosphorylation sites. We have combined the two differentcoloured AlphaScreen® and AlphaLISA® beads into a single-well assay that allows for simultaneous monitoring ofphosphorylation of a given substrate and its binding to themodifying enzyme. As proof-of-concept, we observeddissociation of ERK2 upon phosphorylation by upstream MEK1;whereas upstream (Raf1-MEK1) and downstream (ERK2-Elk-1)kinase-substrate pairs produced distinct phosphorylation-interaction patterns. This technology was further used tocompare selectivity and mechanism of action of three MAPkinase phosphatases on ERK2 and P38α. Finally, a generic ATPsite competitor and an allosteric inhibitor resulted in distinctassay signature patterns when probed on MEK1-ERK2. This dualtechnology can thus be used prior to screening to obtainsignificant biochemical insight as well as in secondary orcounter-screens to discriminate between catalytic sitecompetitors and allosteric modulators.

Introduction2

MAP kinase pathways are activated by numerous stimuli which triggersequential phosphorylation of cognate MAP3K, MAP2K and MAP kinase.Negative regulation namely occurs by dual-specificity MAP kinasephosphatases (MKPs) which catalyze the dephosphorylation of the MAP kinaseactivating residues. In the ERK pathway, Raf isoforms phosphorylate MEK1/2,which in turn activate ERK1/2. The latter can translocate to the nucleus andphosphorylate transcription factors such as Elk-1. All of these enzymaticevents are tightly regulated by protein-protein interaction, some of thebinding interfaces shared by multiple effectors and regulators. For example,MEK1 and MKP-2 use the same docking sites on ERK2 in order tophosphorylate or dephosphorylate it, respectively. Although both inhibitors affected unactive MEK1 and ERK2 binding, their

active MEK1 inhibition mechanism greatly differs. The promiscuous ATPcompetitor staurosporine perturbs ERK2 phosphorylation while partiallyrescuing its interaction with MEK1. In contrast, U0126 displays allostericeffects on MEK1.

Methods3

MEK1 - ERK2 phosphorylation-interaction assay.

Active His-tagged MEK1 was incubated with unphosphorylatedGST-ERK2 in the presence of increasing ATP concentrations.

Regulation of the ERK MAP kinase pathay

Phosphorylation cascade and sequential activation of the MAP3K Raf-1, the MAP2K MEK1 , the MAP kinase ERK2 and the transcription factor Elk-1. MKPs dephosphorylateMAP kinase activating residues.

Pharmacological inhibition of MEK1-ERK2 phosphorylation-interaction

His­MEK1 variants and unactive GST­ERK2 were incubated with increasingamounts of pharmacological kinase inhibitors, in the absence (left panels) orpresence of 10 µM ATP (right panels).

We have combined the flexibility of the AlphaScreen and AlphaLISA platformsinto a single assay enabling the simultaneous monitoring of substratephosphorylation and enzyme interaction, and used the ERK MAP kinasepathway as a model to study these linked but distinct phenomena.

Raf-1 – MEK1 Phosphorylation-

Interaction Assay

5

MEK1 – ERK2 Phosphorylation-

Interaction Assay

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Raf-1 – MEK1 phosphorylation-interaction assay

Active GST-tagged Raf-1 was incubated with unphosphorylatedHis-MEK1 in the presence of increasing ATP concentrations.

Raf-1 – MEK1 interactions can be detected with an antibody-free AlphaLISAset­up, and MEK1 phosphorylation is detected by a dual phosphorylation-specific antibody captured by Protein A AlphaScreen beads. MEK1 directlydissociates from Raf-1 upon phosphorylation.

Phosphorylation and interaction curves for different kinase-substrate pairsgenerate distinct and characteristic pattern.

ERK2 – Elk-1 phosphorylation-interaction assay

Active His-tagged ERK2 was incubated with unphosphorylatedGST-Elk-1 in the presence of increasing ATP concentrations.

MAP Kinase Phosphatase

Selectivity and Mechanism of Action

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All assays were performed in 384 well plates (PerkinElmer) at 23°C in kinasebuffer containing 20 mM Tris pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1mM DTTand 0.01% Tween20. All kinases and substrates were used at 100 nM exceptfor the ERK2-Elk-1 (20 nM each). Kinase reactions were started by adding ATPand incubated for 2h before addition of detection reagents. For phosphataseassays 100 nM of phosphorylated ERK2 were incubated with increasing MKPamounts for 15 min. For all assays, Donor beads were used at 40 µg/mL andAlphaScreen and AlphaLISA Acceptor at 20 µg/mL each. Assays were readtwice by unmodified EnVision readers, first with a dysprosium 572 nm narrowbandwidth filter, then with a europium 615 nm narrow bandwidth filter.

Name Enzymatic class FamilyPhosphorylation

stateActivity Tag Provider

Elk-1 (ΔDNA binding domain) Transcription factor Ets-1 Unphosphorylated Unactive N-GST Cell Signaling

ERK2 Kinase CGMC Phosphorylated Active N-His Sigma

ERK2 Kinase CGMC Unphosphorylated Unactive N-GST Carna Bio

MEK1 Kinase STE Phosphorylated Active N-His Carna Bio

MEK1 Kinase STE Unphosphorylated Unactive N-His SignalChem

MKP-2/DUSP4 Phosphatase Dual specificity Unphosphorylated Active N-His BioMol

MKP-6/DUSP14 Phosphatase Dual specificity Unphosphorylated Active N-His BioMol

MKP-7/DUSP16 Phosphatase Dual specificity Unphosphorylated Active N-His BioMol

Raf-1 (kinase domain) Kinase TKL Mutant Active N-GST Carna Bio

P38α Kinase CGMC Unphosphorylated Unactive N-GST Carna Bio

Recombinant protein kinases were purchased as listed below.Phosphorylation-specific antibodies were purchased from Cell SignalingTechnology; Staurosporine and U0126 from EMD. AlphaScreen and AlphaLISAbeads as well as the EnVision® 2103 reader and filters were fromPerkinElmer.

Dephosphorylation and interaction curves for different phosphatase-substratepairs also generate distinct and characteristic patterns.

MKP – MAP kinase dephosphorylation-interaction assay

Increasing concentrations of His-tagged MKP-2, MKP-6 or MKP-7 were incubated with phosphorylated GST-tagged ERK2 or P38 .

Elk-1 phosphorylation on serine 383 precedes dissociation from ERK2.