High-resolution mapping of prostaglandin E2-dependent signaling networks identifies a constitutively active PKA...

To analyze prostaglandin E(2) (PGE(2)) signaling in lymphoid cells, we introduce a multipronged strategy, combining temporal quantitative phosphoproteomics and phospho flow cytometry. We describe the PGE(2)-induced phosphoproteome by simultaneous monitoring of approximately 250 regulated phospho-epitopes, which, according to kinase prediction algorithms, originate from a limited number of kinase networks. Assessing these signaling pathways by phospho flow cytometry provided higher temporal resolution at various PGE(2) concentrations in multiple lymphoid cell subsets. This showed elevated levels of protein kinase A (PKA) signaling in unstimulated CD8(+)CD45RO(+) T cells, which correlated with suppressed proximal T-cell receptor signaling, indicating that PKA sets the threshold for activation. The combination of phosphoproteomics and high throughput phospho flow cytometry applied here provides a comprehensive generic framework for the analysis of signaling networks in mixed cell populations.