Quantitative Analysis of Phosphotyrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells

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-γ, 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-γ/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and their effects on downstream components including MAPKs.

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

“…Even an increase in phosphorylation of Tyr 505 along with an increase in Lck kinase activity upon CD4 crosslinking was reported (40). This finding is supported by a recent phosphoproteomics study where CD3 crosslinking increased pTyr 505 3-fold over control while pTyr 394 content was nearly unaffected (41). Our data demonstrating a steady Lck conformation throughout T cell activation indicate that neither modulation of Tyr 505 nor Tyr 394 seem to play a dominant role during TCR triggering.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…Phosphorylation of PKC at Tyr-313 by Src family kinases has also been reported in a variety of cell types in response to non-apoptotic stimuli [117][118][119]46,120,32,121,100]. While some of these studies have shown that phosphorylation at Tyr-313 is required for PKC function, one consequence of Tyr-313 phosphorylation is that it regulates the ability of the enzyme to autophosphorylate its own A-loop and thus fine-tune substrate specificity [32,46].…”

Interaction of the Wiskott–Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells

“…Recent data showing levels of WASp tyrosine phosphorylation (and, by extension, activation) to be higher in CD28-than in TCR-stimulated T cells (43), suggest that CD28 actin cytoskeletal effects may be realized via WASp. This possibility is consistent with the severe impairment in CD28 internalization engendered by WASp deficiency and with previous data implicating WASp, NWASp, and other actin regulatory proteins in endocytic processes (19,44).…”