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Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks
BACKGROUND: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of protein...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146539/ https://www.ncbi.nlm.nih.gov/pubmed/21829558 http://dx.doi.org/10.1371/journal.pone.0022928 |
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author | Deswal, Sumit Schulze, Anna K. Höfer, Thomas Schamel, Wolfgang W. A. |
author_facet | Deswal, Sumit Schulze, Anna K. Höfer, Thomas Schamel, Wolfgang W. A. |
author_sort | Deswal, Sumit |
collection | PubMed |
description | BACKGROUND: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success. METHODOLOGY/PRINCIPAL FINDINGS: We use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally. CONCLUSIONS/SIGNIFICANCE: The quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network. |
format | Online Article Text |
id | pubmed-3146539 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-31465392011-08-09 Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks Deswal, Sumit Schulze, Anna K. Höfer, Thomas Schamel, Wolfgang W. A. PLoS One Research Article BACKGROUND: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success. METHODOLOGY/PRINCIPAL FINDINGS: We use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally. CONCLUSIONS/SIGNIFICANCE: The quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network. Public Library of Science 2011-07-29 /pmc/articles/PMC3146539/ /pubmed/21829558 http://dx.doi.org/10.1371/journal.pone.0022928 Text en Deswal 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 Deswal, Sumit Schulze, Anna K. Höfer, Thomas Schamel, Wolfgang W. A. Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks |
title | Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks |
title_full | Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks |
title_fullStr | Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks |
title_full_unstemmed | Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks |
title_short | Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks |
title_sort | quantitative analysis of protein phosphorylations and interactions by multi-colour ip-fcm as an input for kinetic modelling of signalling networks |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146539/ https://www.ncbi.nlm.nih.gov/pubmed/21829558 http://dx.doi.org/10.1371/journal.pone.0022928 |
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