title: 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.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Beck
authors.affiliation: Department of Chemistry, Lincoln University , Lincoln University, Pennsylvania 19352, United States.
authors.forename: Antoneal
authors.initials: A
authors.lastname: Lawrence
authors.affiliation: Department of Chemistry, Lincoln University , Lincoln University, Pennsylvania 19352, United States.
authors.forename: Amar S
authors.initials: AS
authors.lastname: Tung
authors.affiliation: Department of Biochemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
authors.forename: Edward N
authors.initials: EN
authors.lastname: Harris
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004195:Disease Models, Animal; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D065626:Non-alcoholic Fatty Liver Disease / Q000201:enzymology; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D055748:SOXB1 Transcription Factors / Q000737:chemistry
chemical_list: D055748:SOXB1 Transcription Factors; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1