Abstract
In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time. Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E2 (PGE2) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i.e. 98%) contained the consensus motif [R/K][R/K/X]X[pS/pT]. When our data were compared with a large-scale Jurkat T-lymphocyte phosphoproteomics dataset containing more than 10,500 phosphosites, a minimal overlap of 0.2% was observed. This stresses the need for such targeted analyses when the interest is in a particular kinase. Our data provide a resource of likely substrates of PKA, and potentially some substrates of closely related kinases. Network analysis revealed that about half of the observed substrates have been implicated in cAMP-induced signaling. Still, the other half of the here-identified substrates have been less well characterized, representing a valuable resource for future research.
Highlights
From ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; §Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands; ¶Cell Signaling Technology, Danvers, Massachusetts 01923
Qualitative Analysis of prostaglandin E2 (PGE2)-related Phosphorylation in Jurkat T Cells—Here we evaluated and applied a kinasedirected targeted immune-affinity-based proteomics approach to investigate the role of cyclic adenosine monophosphate (cAMP)/protein kinase (PKA) signaling downstream of PGE2 in Jurkat T cells
We concluded that PGE2 stimulus (10 M) induced a rapid, maximal increase in the phosphorylation of serine/threonine residues with arginine at the -3 position after only 1 min
Summary
Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach*□S. Boersema et al [28] introduced an alternative sensitive and accurate triplex labeling approach using inexpensive reagents (i.e. formaldehyde) that is much less limited in terms of the sample type or amount We combined this latter stable-isotope dimethyl labeling approach [27,28,29] with highly specific antibodies raised against a set of cAMP-dependent protein kinase (PKA) phosphorylated substrates as based on the current literature (11, 30 –34). We efficiently enriched close to 650 phosphopeptides containing the [R/K][R/K/X]X[pS/pT] consensus motif Almost all these sites were absent in a recently reported comprehensive phosphoproteomics dataset of Jurkat T cells [12], compiled using shotgun strong cation exchange–immobilized metal ion affinity chromatography analysis and containing ϳ10,500 phosphorylation sites, illustrative of the complementarity and selectivity of our approach. The dataset presented here can be considered as a comprehensive and reliable resource for future research into cAMPrelated signaling
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