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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

Paper ID Volume ID Publish Year Pages File Format Full-Text
30303 44468 2014 9 PDF Available
Title
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
Abstract

•Protein phosphatase complexes are isolated by surface plasmon resonance based binding technique.•Biotin–microcystin-LR (MC-LR) coated sensorchip surface is prepared.•This surface is suitable to capture protein phosphatases and their binding proteins.•Protein phosphatase-1 and -2A complexes from HaCaT cells are characterized.•From UVA-irradiated cells less phosphatases are recovered than from untreated cells.

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.

Keywords
Protein phosphatase-1 and -2A; Microcystin-LR; Biotin–microcystin-LR conjugate; Surface plasmon resonance; Protein phosphatase interacting proteins; HaCaT cells
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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
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Photochemistry and Photobiology B: Biology - Volume 138, 5 September 2014, Pages 240–248
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us