High-resolution mapping of prostaglandin E2–dependent signaling networks identifies a constitutively active PKA signaling node in CD8+CD45RO+ T cells

Abstract

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

A PKA-ezrin-connexin 43 signaling complex controls gap junction communication and thereby trophoblast cell fusion

“…Pull-down assays cAMP pulldown assays (8-AHA-cAMP-agarose beads) were performed as described previously (Oberprieler et al, 2010;Scholten et al, 2006). Cell lysate (500 mg proteins) was incubated with purified RI-FLAG or RII-FLAG proteins (100 mg).…”

T Cell-Signaling Network Analysis Reveals Distinct Differences between CD28 and CD2 Costimulation Responses in Various Subsets and in the MAPK Pathway between Resting and Activated Regulatory T Cells

“…However, recent technical developments, including fluorescent cell barcoding (FCB) (31) and a growing number of phospho-epitope-specific Abs, have made it possible to use phospho-epitope-specific flow (phospho-flow) cytometry-based methods to study signaling processes at single-cell resolution in several phenotypically defined T cell subsets simultaneously (32)(33)(34). Furthermore, these developments have enabled an increase in the resolution to a level at which signaling differences can be linked to functional properties in small subsets of cells.…”

“…Three-dimensional FCB was carried out, as previously described (34,35). In brief, fixed cells were incubated with varying concentrations of esters conjugated to Pacific Blue (100, 25, 6.3, 0.7 pg/ml; Molecular Probes, Invitrogen), Pacific Orange (1000, 250, 41.7, 4.2 pg/ml; Molecular Probes, Invitrogen), and Alexa Fluor 488 (50, 12.5, 3.1, 0.3 pg/ml; Molecular Probes, Invitrogen) for 20 min at room temperature.…”

Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach

“…The combination of phosphopeptide enrichment (3)(4)(5)(6), stable isotope labeling, and high-resolution mass spectrometry (MS) methods (7)(8)(9) has become the method of choice for the identification of novel phosphorylation sites and for the quantitation of temporal dynamics within signaling networks (10,11), allowing the behavior of thousands of phosphorylation sites to be studied in a single experiment (10,12,13). Nowadays, one of the most commonly adopted high-throughput phosphoproteomics strategies utilizes two consecutive separation steps: (i) an initial fractionation to reduce the sample complexity, and (ii) a phosphopeptide-specific affinity purification.…”