Elucidating missing links of the TCR signaling network

Just published:
Phosphorylation site dynamics of early T-cell receptor signaling. LA Chylek, V Akimov, J Dengjel,  KTG Rigbolt, WS Hlavacek, B Blagoev. PLOS ONE 9, e104240

Stimulation of the T-cell receptor (TCR) can trigger a cascade of biochemical signaling events with far-reaching consequences for the T cell, including changes in gene regulation and remodeling of the actin cytoskeleton. A driving force in the initiation of signaling is phosphorylation and dephosphorylation of signaling proteins. This process has been difficult to characterize in detail because phosphorylation takes place rapidly, on the timescale of seconds, which can confound efforts to decode the order in which events occur. In addition, multiple residues in a protein may be phosphorylated, each involved in distinct regulatory mechanisms, necessitating analysis of individual sites.

To characterize the dynamics of site-specific phosphorylation in the first 60 seconds of TCR signaling, we stimulated cells for precise lengths of time using a quench-flow system and quantified changes in phosphorylation using mass spectrometry-based phosphoproteomics. We developed a computational model that reproduced experimental measurements and generated predictions that were validated experimentally. We found that the phosphatase SHP-1, previously characterized primarily as a negative regulator, plays a positive role in signal initiation by dephosphorylating negative regulatory sites in other proteins. We also found that the actin regulator WASP is rapidly activated via a shortcut pathway, distinct from the longer pathway previously considered to be the main route for WASP recruitment. Through iterative experimentation and model-based analysis, we have found that early signaling may be driven by transient mechanisms that are likely to be overlooked if only later timepoints are considered.