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.
authors.affiliation: MTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Debrecen, Hungary; Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary.
authors.forename: Bálint
authors.initials: B
authors.lastname: Bécsi
authors.affiliation: Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary.
authors.forename: Dóra
authors.initials: D
authors.lastname: Dedinszki
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen, Hungary.
authors.forename: Gyöngyi
authors.initials: G
authors.lastname: Gyémánt
authors.affiliation: Department of Botany, University of Debrecen, Debrecen, Hungary.
authors.forename: Csaba
authors.initials: C
authors.lastname: Máthé
authors.affiliation: Department of Botany, University of Debrecen, Debrecen, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Vasas
authors.affiliation: Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary.
authors.forename: Beáta
authors.initials: B
authors.lastname: Lontay
authors.affiliation: MTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Debrecen, Hungary; Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary. Electronic address: erdodi@med.unideb.hu.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Erdődi
mesh_terms: D000906:Antibodies / Q000276:immunology; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D008387:Marine Toxins; D052998:Microcystins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014466:Ultraviolet Rays
chemical_list: D000906:Antibodies; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D052998:Microcystins; D021122:Protein Subunits; D034741:RNA, Small Interfering; D001710:Biotin; D019809:Streptavidin; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; C057862:cyanoginosin LR
keywords: Biotin–microcystin-LR conjugate; HaCaT cells; Microcystin-LR; Protein phosphatase interacting proteins; Protein phosphatase-1 and -2A; Surface plasmon resonance