Abstract
BackgroundTo 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 FindingsWe 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/SignificanceThe 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.
Highlights
Inducible protein-protein interactions and post-translational modifications are the major mode of cellular communication and are responsible for cellular decisions such as cell proliferation, differentiation, survival or death [1,2]
The mean fluorescence intensities (MFIs) of the anti-ZAP70 and anti-pY319ZAP70 stains were normalized by the MFI of the anti-CD3e stain (Fig. 1bc)
We improved one-colour immunoprecipitation measured by flow cytometry (IP-flow cytometry (FCM)) to develop a powerful technology platform for the high-throughput generation of multidimensional and quantitative data that serves as a new input for quantitative analysis
Summary
Inducible protein-protein interactions and post-translational modifications are the major mode of cellular communication and are responsible for cellular decisions such as cell proliferation, differentiation, survival or death [1,2]. Despite their importance, precise quantitative measurements of these interactions and modifications remain difficult. The most commonly used protein analysis method is immuno-precipitation (IP) followed by SDSPAGE and Western blotting (IP-WB) This technique has provided invaluable insight into signalling pathways; it is neither very quantitative nor high-throughput. Precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
