Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors

Abstract

We introduce a versatile approach for repurposing protein kinase chemosensors, containing the phosphorylation-sensitive sulfonamido-oxine fluorophore termed Sox, for the specific determination of endogenous protein phosphatase activity from whole cell lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for protein tyrosine phosphatase-1B (PTP1B), an established signaling node in human disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) full-length recombinant PTP1B and is remarkably selective for PTP1B among a panel of highly homologous tyrosine phosphatases. Coupling this robust activity probe with the specificity of antibodies allowed for the temporal analysis of endogenous PTP1B activity dynamics in lysates generated from HepG2 cells after stimulation with insulin. Lastly, we leveraged this assay format to profile PTP1B activity perturbations in a rat model of nonalcoholic fatty liver disease (NAFLD), providing direct evidence for elevated PTP1B catalytic activity in this disease state. Given the modular nature of this assay, we anticipate that this approach will have broad utility in monitoring phosphatase activity dynamics in human disease states.

Interrogating Protein Phosphatases with Chemical Activity Probes

“…[68] In this strategy, phosphorylated Sox-containing peptides are synthesized and produce fluorescence through chelation-enhanced fluorescence (CHEF) with magnesium. Upon dephosphorylation, fluorescence is reduced at a rate that is proportional to PP activity ( 41 , Figure 9).…”

2016 EMBO Chemical Biology Conference

“…As examples, both caged luciferins and split luciferase were used to form the basis of cellular-proximity sensors. [39] Jon Beck, as tudent in Cliff Stain's lab (Universityo fN ebraska-Lincoln) presented his work on sensors of tyrosine phosphatase activity with picomolar limits of detection [40] and Manuela Zaccolo (University of Oxford) discussed novel FRET-basedr eporterst hat detect compartmentalised cAMP with unprecedented spatialr esolution. Dmytro Yushchenko from Carsten Schultz's lab (EMBL Heidelberg) introduced tools for studying lipid signalling such as caged arachidonic acidd erivatives and photoswitchable diacylglycerols (DAGs).…”

Design and synthesis of fluorescent activity probes for protein phosphatases

“…1) (Pearce, Jotterand, Carrico, & Imperiali, 2001;Shults, Pearce, & Imperiali, 2003). This strategy can be applied to both protein tyrosine phosphatases (PTPs) (Beck, Lawrence, et al, 2016) and protein serine/threonine phosphatases (PSPs) (Beck, Truong, et al, 2016). Furthermore, if sufficiently selective substrate sequences can be identified, PP activity can be monitored in unfractionated cell lysates providing insight into the temporal dynamics of PP signaling under relevant biological stimuli (Beck, Truong, et al, 2016).…”