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Updated: Aug 11, 2026
Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells
Published on: September 9, 2021
Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in Jurkat cells
1Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
The mechanism by which stimulation of coreceptors such as CD28 contributes to full activation of TCR signaling pathways has been intensively studied, yet quantitative measurement of costimulation effects on functional TCR signaling networks has been lacking. In this study, phosphotyrosine networks triggered by CD3, CD28, or CD3 and CD28 costimulation were analyzed by site-specific quantitative phosphoproteomics, resulting in identification of 101 tyrosine and 3 threonine phosphorylation sites and quantification of 87 sites across four cell states. As expected, CD3 stimulation induced phosphorylation of CD3 chains and upstream components of TCR pathways such as Zap70, while CD28 stimulation induced phosphorylation of CD28, Vav-1, and other adaptor proteins including downstream of tyrosine kinase 1, Grb2-associated protein 2 (Grap2), and Wiskott-Aldrich syndrome protein. CD3 and CD28 costimulation induced a complex response including decreased threonine phosphorylation in the ERK1 and ERK2 activation loops and increased phosphorylation of selected tyrosine sites on ERK1/2, p38, phospholipase C-gamma, Src homology 2 domain-containing transforming protein 1, Grap2, and Vav-1, potentiating T cell activation. Hierarchical clustering and self-organizing maps were used to identify modules of coregulated phosphorylation sites within the network. Quantitative information in our study suggests quantitative and qualitative contribution by costimulation of CD28 on CD3-stimulated TCR signaling networks via enhanced phosphorylation of phospholipase C-gamma/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and their effects on downstream components including MAPKs.
Insights
T-cell activation involves complex signaling pathways. This study quantifies how CD28 costimulation enhances T-cell receptor (TCR) signaling networks, revealing key phosphorylation events that boost T-cell responses.
Area of Science:
- Immunology
- Cell Signaling
- Proteomics
Background:
- T-cell activation relies on T-cell receptor (TCR) signaling, modulated by costimulatory molecules like CD28.
- Quantitative insights into how CD28 costimulation impacts TCR signaling networks have been limited.
Purpose of the Study:
- To quantitatively analyze phosphotyrosine networks in T-cells upon CD3, CD28, or combined CD3/CD28 stimulation.
- To elucidate the specific contributions of CD28 costimulation to TCR signaling pathway activation.
Main Methods:
- Site-specific quantitative phosphoproteomics was employed to profile phosphorylation sites.
- Hierarchical clustering and self-organizing maps were used to analyze signaling modules.
Main Results:
- Identified 101 tyrosine and 3 threonine phosphorylation sites, quantifying 87 sites.
- CD3 stimulation activated TCR pathway components; CD28 stimulation activated adaptor proteins.
- CD3/CD28 costimulation induced complex changes, including enhanced phosphorylation of key signaling proteins (e.g., PLCγ, SHIP1, Grap2, Vav-1) and MAPKs, potentiating T-cell activation.
Conclusions:
- CD28 costimulation quantitatively and qualitatively enhances CD3-induced TCR signaling networks.
- Enhanced phosphorylation of specific signaling nodes by CD28 significantly impacts downstream T-cell activation pathways.
