Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

Abstract

Activation of the T-cell receptor (TCR) and that of the B-cell receptor (BCR) elicits tyrosinephosphorylation of proteins that belongs to similar functional categories, but result in distinct cellular responses. Large-scale analyses providing an overview of the signaling pathways downstream of TCR or BCR have not been described, so it has been unclear what components of these pathways are shared and which are specific. We have now performed a systematic analysis and provide a comprehensive list of tyrosine-phosphorylated proteins (PY proteome) with quantitative data on their abundance in T cell, B cell, and nonlymphoid cell lines. Our results led to the identification of novel tyrosine-phosphorylated proteins and signaling pathways not previously implicated in immunoreceptor signal transduction, such as clathrin, zonula occludens 2, eukaryotic translation initiation factor 3, and RhoH, suggesting that TCR or BCR signaling may be linked to downstream processes such as endocytosis, cell adhesion, and translation. Thus comparative and quantitative studies of tyrosine-phosphorylation have the potential to expand knowledge of signaling networks and to promote understanding of signal transduction at the system level.

A Unique Carboxyl-terminal Insert Domain in the Hematopoietic-specific, GTPase-deficient Rho GTPase RhoH Regulates Post-translational Processing

“…This suggests that wtRhoH interacts with chaperone molecules such as Hsp90 that protect it from degradation via the CMA pathway and that reduced RhoH protein levels demonstrated after treatment with GA result from lack of protection from degradation due to disruption of chaperone complexes and activation of CMA. Of note, in in vivo biotinylation experiments we have identified members of the heat shock protein family including Hsp90 and Hsp70 as novel interaction partners of RhoH, 3 which is in keeping with our current findings and previous reports (49). The absence of the insert sequence LFSINE appears to retard RhoH degradation via CMA, suggesting that LFSINE may be needed as a recognition signal for lysosomal uptake and degradation.…”

Swiprosin-1/EFhd2 Controls B Cell Receptor Signaling through the Assembly of the B Cell Receptor, Syk, and Phospholipase C γ2 in Membrane Rafts

“…Drosophila Swip-1 is expressed in larvae from stage 9 on in the ventral head mesoderm and at stage 10 in hemocytes of the head mesoderm (75). In analogy, Swip-1 is expressed in cells originating from the mesoderm in mice and man, namely B and T lymphocytes, NK-like cells, and the monocytic cell line RAW264.7 (22,54,76,77). Swip-1 becomes upregulated upon stimulation of RAW264 cells with receptor activator for NF-kB ligand and TNF-a, conditions that promote inflammation and osteoclast differentiation (77).…”

Tools used to study how protein complexes are assembled in signaling cascades

“…The method comprises of overexpressing a dual-tagged target protein in host cells, isolation of the fusion protein using two binding steps and then identifying co-bound proteins by MS. 1,[87][88][89][90] Because two tags are expressed with the protein, there are lower levels of non-specific binding and increased complex recovery. The system also benefits from being compatible with several protein identification processes such as native-PAGE, 2D-electrophoresis, protein gel blotting and MS. [91][92][93] Limitations in the process include loss of weakly bound interacting proteins and there is always the possibility that the tag will interfere with folding and function of the protein disrupting how the protein interacts with other proteins.…”