Quantitative Phosphoproteomic Analysis of T Cell Receptor Signaling Reveals System-Wide Modulation of Protein-Protein Interactions

Abstract

Protein phosphorylation events during T cell receptor (TCR) signaling control the formation of complexes among proteins proximal to the TCR, the activation of kinase cascades, and the activation of transcription factors; however, the mode and extent of the influence of phosphorylation in coordinating the diverse phenomena associated with T cell activation are unclear. Therefore, we used the human Jurkat T cell leukemia cell line as a model system and performed large-scale quantitative phosphoproteomic analyses of TCR signaling. We identified 10,665 unique phosphorylation sites, of which 696 showed TCR-responsive changes. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation: patterning of surface proteins, endocytosis of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA. Further, case-by-case analysis of TCR-responsive phosphorylation sites on proteins belonging to relevant functional modules together with network analysis allowed us to deduce that serine-threonine (S-T) phosphorylation modulated protein-protein interactions (PPIs) in a system-wide fashion. We also provide experimental support for this inference by showing that phosphorylation of tubulin on six distinct serine residues abrogated PPIs during the assembly of microtubules. We propose that modulation of PPIs by stimulus-dependent changes in S-T phosphorylation state is a widespread phenomenon applicable to many other signaling systems.

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

“…Indeed, recent studies showed that O-GlcNAc glycosylation of nuclear pore proteins prevents their proteasomal degradation (85) and facilitates the transport of protein cargo (86). Interestingly, phosphorylation of nuclear pore proteins, including NUP214, also increases after T cell activation (83), which raises the possibility of multiple posttranslational modifications working synergistically to regulate nuclear pore function in activated T cells.…”

Database independent proteomics analysis of the ostrich and human proteome

“…In all these examples, a traditional database search would have had difficulty resolving the correct peptides. The phosphorylated ostrich peptide sequences, although conserved in human (34) are completely absent from the IPI chicken database and the lysine acetylated peptides from ostrich contain point mutations when compared to their chicken and other homolog sequences. Moreover, the second acetylated peptide (KQVVDSAYEVNKacL) contains additionally a unique, single nucleotide exchange, isoleucine to asparagine mutation (indicated in green, Fig.…”

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

“…Phosphoproteomics analysis has also revealed that PKC undergoes phosphorylation at Thr-685 between the TM and HM sites in response to TCR-stimulation [101]. The impact of Thr-685 phosphorylation on PKC function in T cells remains to be addressed.…”