Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

Abstract

, and the nucleus. The extracts contain all major classes of protein phosphatases and catalyze dephosphorylation of plate-bound phosphosubstrates in a microtiter format, with cellular activity quantified at the end point by phosphospecific ELISA. The platform is optimized for six phosphosubstrates (ERK2, JNK1, p38␣, MK2, CREB, and STAT1) and measures specific activities from extracts of fewer than 50,000 cells. The assay was exploited to examine viral and antiviral signaling in AC16 cardiomyocytes, which we show can be engineered to serve as susceptible and permissive hosts for CVB3. Phosphatase responses were profiled in these cells by completing a full-factorial experiment for CVB3 infection and type I/II interferon signaling. Over 850 functional measurements revealed several independent, subcellular changes in specific phosphatase activities. During CVB3 infection, we found that type I interferon signaling increases subcellular JNK1 phosphatase activity, inhibiting nuclear JNK1 activity that otherwise promotes viral protein synthesis in the infected host cell. Our assay provides a high-throughput way to capture perturbations in important negative regulators of intracellular signaltransduction networks. Molecular & Cellular Proteomics 16:

An ultrasensitive fiveplex activity assay for cellular kinases

“…6) and may reflect samplespecific adaptations to CVB3 (Ref. 13 ). One potential mechanism involves autocrine signaling circuits, which are variably time-evolving and iterative 45 .…”

“…Linear response characteristics obviate the need for logistic curve fitting of standards to make quantitative measurements 51 . We repeated the titration with extracts from AC16-CAR cells, a human ventricular cardiomyocyte cell line 52 engineered to express the coxsackievirus and adenovirus receptor 13 . When using AC16-CAR extracts, we again observed linear behavior for all five kinases in with R 2 values of 0.94 or better (Supplemental Fig.…”

“…As a representative cellular perturbation to examine with the fiveplex assay, we selected acute infection by the picornavirus CVB3. During transmission and pathogenesis, CVB3 infects multiple cell types 63 , including the gut epithelium (e.g., HT29 cells 64 ) and cardiomyocytes (e.g., AC16-CAR cells 13 ). In other model cell systems, CVB3 impacts all five kinases in the assay [65][66][67][68] .…”

“…Wells were washed twice with 200 µL 50 mM Tris (pH 7.5), 150 mM NaCl and twice with 200 µL 30 mM Tris (pH 7.5), 22.5 mM MgCl2, 7.5 mM β-glycerophosphate, 1.5 mM EGTA, 300 µM sodium orthovanadate, 300 µM dithiothreitol before eluting with 50 µL of 62.5 mM Tris-HCL (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromophenol blue, 100 mM dithiothreitol and incubating at 100ºC. Immunoblotting of immunoprecipitated proteins was performed as described 13 with the following primary antibodies: IKKα (1:1000, Cell Signaling Technology #2682) and IKKβ (1:1000, Cell Signaling Technology #8943). Both species were detected with IrDye800conjugated anti-rabbit secondary (Licor #926-32211).…”

The Host-Pathogen Ecosystem Viewed Through the Prism of Proteomics

“…To provide a robust, high-throughput platform for studying the activity of key phosphatases in innate immune response, Janes and colleagues describe a method integrating on-plate cellular fractionation, isolation of several phosphatases (e.g. STAT1, ERK2, JNK1), and quantification of enzymatic activity by ELISA (17). They demonstrate the usefulness of this method for monitoring relative phosphatase activity in specific subcellular compartments in response to infection of cardiomyocytes with coxsackievirus B3.…”