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

Bálint Bécsi, Dóra Dedinszki, G. Gyémánt, Csaba Máthé, G. Vasas, Beáta Lontay, F. Erdődi

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)240-248
Number of pages9
JournalJournal of Photochemistry and Photobiology, B: Biology
Volume138
DOIs
Publication statusPublished - Sep 5 2014

Fingerprint

biotin
phosphatases
Surface Plasmon Resonance
Phosphoprotein Phosphatases
Biotin
surface plasmon resonance
Phosphoric Monoester Hydrolases
proteins
cells
molecules
Proteins
Catalytic Domain
Streptavidin
inhibitors
Myosin-Light-Chain Phosphatase
Protein Phosphatase 1
Protein Phosphatase 2
cyanoginosin LR
myosins
Confocal Microscopy

Keywords

  • Biotin-microcystin-LR conjugate
  • HaCaT cells
  • Microcystin-LR
  • Protein phosphatase interacting proteins
  • Protein phosphatase-1 and -2A
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Radiation
  • Radiology Nuclear Medicine and imaging
  • Radiological and Ultrasound Technology
  • Biophysics
  • Medicine(all)

Cite this

@article{8a8dd24eb342476180afaf4b18031a77,
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 = "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 = "Biotin-microcystin-LR conjugate, HaCaT cells, Microcystin-LR, Protein phosphatase interacting proteins, Protein phosphatase-1 and -2A, Surface plasmon resonance",
author = "B{\'a}lint B{\'e}csi and D{\'o}ra Dedinszki and G. Gy{\'e}m{\'a}nt and Csaba M{\'a}th{\'e} and G. Vasas and Be{\'a}ta Lontay and F. Erdődi",
year = "2014",
month = "9",
day = "5",
doi = "10.1016/j.jphotobiol.2014.06.004",
language = "English",
volume = "138",
pages = "240--248",
journal = "Journal of Photochemistry and Photobiology B: Biology",
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TY - JOUR

T1 - 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

AU - Bécsi, Bálint

AU - Dedinszki, Dóra

AU - Gyémánt, G.

AU - Máthé, Csaba

AU - Vasas, G.

AU - Lontay, Beáta

AU - Erdődi, F.

PY - 2014/9/5

Y1 - 2014/9/5

N2 - 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.

AB - 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.

KW - Biotin-microcystin-LR conjugate

KW - HaCaT cells

KW - Microcystin-LR

KW - Protein phosphatase interacting proteins

KW - Protein phosphatase-1 and -2A

KW - Surface plasmon resonance

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U2 - 10.1016/j.jphotobiol.2014.06.004

DO - 10.1016/j.jphotobiol.2014.06.004

M3 - Article

C2 - 24993084

AN - SCOPUS:84903717145

VL - 138

SP - 240

EP - 248

JO - Journal of Photochemistry and Photobiology B: Biology

JF - Journal of Photochemistry and Photobiology B: Biology

SN - 1011-1344

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