Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors

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作者：

JR Beck，A Lawrence，AS Tung，EN Harris，CI Stains

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摘要：

We introduce a versatile approach for repurposing kinase chemosensors, containing the -sensitive sulfonamido-oxine fluorophore termed , for the specific determination of endogenous activity from whole lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for (), an established node in disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) full-length recombinant and is selective for among a panel of highly homologous tyrosine . Coupling this robust activity probe with the specificity of allowed for the temporal analysis of endogenous activity dynamics in lysates generated from HepG2 after stimulation with . Lastly, we leveraged this assay format to profile activity perturbations in a model of (NAFLD), providing direct evidence for elevated in this disease state. Given the modular nature of this assay, we anticipate that this approach will have broad utility in monitoring activity dynamics in disease states.

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DOI：

10.1021/acschembio.5b00506

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年份：

2016