Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes.
Cellular responses to stimuli involve dynamic and localized changes in protein kinases and phosphatases. Here, we report a generalized functional assay for high-throughput profiling of multiple protein phosphatases with subcellular resolution and apply it to analyze coxsackievirus B3 (CVB3) infection counteracted by interferon signaling. Using on-plate cell fractionation optimized for adherent cells, we isolate protein extracts containing active endogenous phosphatases from cell membranes, the cytoplasm, 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, p38α, 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 signal-transduction networks.
From the ‡Department of Biomedical Engineering.
Millie
M
Shah
From the ‡Department of Biomedical Engineering.
Christian M
CM
Smolko
From the ‡Department of Biomedical Engineering.
Sarah
S
Kinicki
the §Department of Chemistry, and.
Zachary D
ZD
Chapman
the ‖Center for Cell Signaling and Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22908.
David L
DL
Brautigan
From the ‡Department of Biomedical Engineering, kjanes@virginia.edu.
Kevin A
KA
Janes
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003384:Coxsackievirus Infections / Q000378:metabolism* / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000821:virology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction
D010749:Phosphoprotein Phosphatases