Identification of protein phosphatase interacting proteins from normal and UVA-irradiated HaCaT cell lysates by surface plasmon resonance based binding technique using biotin–microcystin-LR as phosphatase capturing molecule

Identification of the interacting proteins of protein phosphatases is crucial to understand the cellular roles of these enzymes. Microcystin-LR (MC-LR), a potent inhibitor of protein phosphatase-1 (PP1), -2A (PP2A), PP4, PP5 and PP6, was biotinylated, immobilized to streptavidin-coupled sensorchip surface and used in surface plasmon resonance (SPR) based binding experiments to isolate phosphatase binding proteins. Biotin–MC-LR captured PP1 catalytic subunit (PP1c) stably and the biotin–MC-LR-PP1c complex was able to further interact with the regulatory subunit (MYPT1) of myosin phosphatase. Increased biotin–MC-LR coated sensorchip surface in the Surface Prep unit of Biacore 3000 captured PP1c, PP2Ac and their regulatory proteins including MYPT1, MYPT family TIMAP, inhibitor-2 as well as PP2A-A and -Bα-subunits from normal and UVA-irradiated HaCaT cell lysates as revealed by dot blot analysis of the recovered proteins. Biotin–MC-LR was used for the subcellular localization of protein phosphatases in HaCaT cells by identification of phosphatase-bound biotin–MC-LR with fluorescent streptavidin conjugates. Partial colocalization of the biotin–MC-LR signals with those obtained using anti-PP1c and anti-PP2Ac antibodies was apparent as judged by confocal microscopy. Our results imply that biotin–MC-LR is a suitable capture molecule in SPR for isolation of protein phosphatase interacting proteins from cell lysates in sufficient amounts for immunological detection.