Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here, we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells and found 168 phosphorylations regulated upon IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ17 cells. IL-23induced RLC phosphorylation required Janus kinase 2 (JAK2) and Rho-associated protein kinase (ROCK) catalytic activity, and further study of the IL-23/ROCK connection revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells through ROCK activation. In addition, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work (i) provides new insights into phosphorylation networks that control Th17 cells, (ii) widely expands the current knowledge on IL-23 signaling, and (iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.

Phosphorylation is the most abundant post-translational modification, regulating several aspects of protein and cell function. Quantitative phosphoproteomics approaches have expanded the scope of phosphorylation analysis enabling the quantification of changes in thousands of phosphorylation sites simultaneously in two or more conditions. These approaches offer a global view of the impact of cellular perturbations such as extracellular stimuli or gene ablation in intracellular signaling networks. Such great potential also brings on a new challenge: to identify, among the thousands of phosphorylations found in global phosphoproteomics studies, the small subset of site-specific phosphorylations expected to be functionally relevant. This review focus on updating and integrating findings on T lymphocyte signaling generated using global phosphoproteomics approaches, drawing attention on the biological relevance of the obtained data.