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Status: Bibliographieeintrag

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Verfasst von:Deswal, Sumit [VerfasserIn]   i
 Schulze, Anna [VerfasserIn]   i
 Höfer, Thomas [VerfasserIn]   i
 Schamel, Wolfgang [VerfasserIn]   i
Titel:Quantitative analysis of protein phosphorylations and interactions by multi-colour IP-FCM as an input for kinetic modelling of signalling networks
Verf.angabe:Sumit Deswal, Anna K. Schulze, Thomas Höfer, Wolfgang W.A. Schamel
Jahr:2011
Umfang:13 S.
Fussnoten:Gesehen am 09.05.2022
Titel Quelle:Enthalten in: PLOS ONE
Ort Quelle:San Francisco, California, US : PLOS, 2006
Jahr Quelle:2011
Band/Heft Quelle:6(2011), 7 vom: Juli, Artikel-ID e22928, Seite 1-13
ISSN Quelle:1932-6203
Abstract: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.
DOI:doi:10.1371/journal.pone.0022928
URL:Bitte beachten Sie: Dies ist ein Bibliographieeintrag. Ein Volltextzugriff für Mitglieder der Universität besteht hier nur, falls für die entsprechende Zeitschrift/den entsprechenden Sammelband ein Abonnement besteht oder es sich um einen OpenAccess-Titel handelt.

kostenfrei: Volltext ; Verlag: https://doi.org/10.1371/journal.pone.0022928
 kostenfrei: Volltext: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022928
 DOI: https://doi.org/10.1371/journal.pone.0022928
Datenträger:Online-Ressource
Sprache:eng
Sach-SW:Cell staining
 Flow cytometry
 Immunoprecipitation
 Latex
 Mathematical models
 Phosphatases
 Phosphorylation
 T cells
K10plus-PPN:1801218897
Verknüpfungen:→ Zeitschrift

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