High-resolution mapping of prostaglandin E2-dependent signaling networks identifies a constitutively active PKA signaling node in CD8+CD45RO+ T cells.
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.
Oberprieler
Nikolaus G
NG
The Biotechnology Centre of Oslo and Centre for Molecular Medicine, Nordic EMBL Partnership, University of Oslo, Blindern, Oslo, Norway.
Lemeer
Simone
S
Kalland
Maria E
ME
Torgersen
Knut M
KM
Heck
Albert J R
AJ
Taskén
Kjetil
K
D000595:Amino Acid Sequence; D017252:CD3 Complex / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D017493:Leukocyte Common Antigens / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D016176:T-Lymphocyte Subsets / Q000187:drug effects* / Q000378:metabolism*
D017252:CD3 Complex; D010748:Phosphopeptides; D020543:Proteome; D011948:Receptors, Antigen, T-Cell; D017868:Cyclic AMP-Dependent Protein Kinases; D017493:Leukocyte Common Antigens; D015232:Dinoprostone