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Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay

Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The a...

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Autores principales: Böhmer, Sylvia-Annette, Weibrecht, Irene, Söderberg, Ola, Böhmer, Frank-D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641115/
https://www.ncbi.nlm.nih.gov/pubmed/23650535
http://dx.doi.org/10.1371/journal.pone.0062871
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author Böhmer, Sylvia-Annette
Weibrecht, Irene
Söderberg, Ola
Böhmer, Frank-D.
author_facet Böhmer, Sylvia-Annette
Weibrecht, Irene
Söderberg, Ola
Böhmer, Frank-D.
author_sort Böhmer, Sylvia-Annette
collection PubMed
description Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP-1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation.
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spelling pubmed-36411152013-05-06 Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay Böhmer, Sylvia-Annette Weibrecht, Irene Söderberg, Ola Böhmer, Frank-D. PLoS One Research Article Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP-1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation. Public Library of Science 2013-05-01 /pmc/articles/PMC3641115/ /pubmed/23650535 http://dx.doi.org/10.1371/journal.pone.0062871 Text en © 2013 Böhmer et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Böhmer, Sylvia-Annette
Weibrecht, Irene
Söderberg, Ola
Böhmer, Frank-D.
Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
title Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
title_full Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
title_fullStr Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
title_full_unstemmed Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
title_short Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
title_sort association of the protein-tyrosine phosphatase dep-1 with its substrate flt3 visualized by in situ proximity ligation assay
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641115/
https://www.ncbi.nlm.nih.gov/pubmed/23650535
http://dx.doi.org/10.1371/journal.pone.0062871
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