Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells

Abstract

Specific functions for different cyclic nucleotide phosphodiesterases (PDEs) have not yet been identified in most cell types. Conventional approaches to study PDE function typically rely on measurements of global cAMP, general increases in cAMP-dependent protein kinase (PKA), or the activity of exchange protein activated by cAMP (EPAC). Although newer approaches using subcellularly targeted FRET reporter sensors have helped define more compartmentalized regulation of cAMP, PKA, and EPAC, they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem, we have begun to use an unbiased mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the functional pools of cAMP/PKA/EPAC that are regulated by specific cAMP-PDEs (the PDE-regulated phosphoproteomes). In Jurkat cells we find multiple, distinct PDE-regulated phosphoproteomes that can be defined by their responses to different PDE inhibitors. We also find that little phosphorylation occurs unless at least two different PDEs are concurrently inhibited in these cells. Moreover, bioinformatics analyses of these phosphoproteomes provide insight into the unique functional roles, mechanisms of action, and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. The data strongly suggest that the phosphorylation of many different substrates contributes to cAMP-dependent regulation of these cells. The findings further suggest that the approach of using selective, inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling.

Different phosphodiesterases (PDEs) regulate distinct phosphoproteomes during cAMP signaling

“…This analysis revealed clusters of interacting proteins associated with particular cell pathways, which differed between the PDE3 and -4 and the PDE1, -7, and -8 inhibitor treatment groups. Finally, Beltejar et This study by Beltejar et al (16) has important clinical implications. Because so many fundamental physiological processes are regulated by cAMP signaling, PDE inhibitors are under intense development for a whole host of disorders and are seeing increasing approval for clinical use.…”

“…Although the use of subcellularly targeted FRET reporter sensors have helped to confirm the role of PDEs in regulating the temporal and spatial control of cAMP during signal transduction, this approach only has limited ability to link specific PDEs to regulation of downstream effector molecules and biological functions. In PNAS, Beltejar et al (16) use mass spectrometry coupled with the use of specific isozyme-selective PDE inhibitors to characterize, for the first time, the phosphoproteomes of functional pools of cAMP regulated by specific PDEs, to delineate which PDEs control phosphorylation of which proteins, leading to regulation of different responses by different PDEs.…”

“…Using CD3/CD28-stimulated Jurkat T leukemic cells, Beltejar et al (16) coupled mass spectrometry phosphoproteomic analyses with treatment using selective inhibitors of PDEs 1, 3, 4, 7, and 8, in the presence and absence of low physiological concentrations of PGE 2 , to characterize the PDE-regulated phosphoproteome of these cells. To determine which sets of PDE inhibitors to use in these studies, the authors first treated Jurkat cells with individual isozymeselective PDE inhibitors or various combinations of them, and measured resulting changes in cAMP levels.…”

“…Beltejar et al (16) carried out several other important bioinformatics analyses. First, by searching a database of phosphosites annotated for predicted regulatory function, the authors were able to identify which phosphosites were most likely to be biologically relevant.…”

Axelrod Symposium 2019: Phosphoproteomic Analysis of G-Protein–Coupled Pathways

“…To confirm the downstream effects and map out the proteins targeted by PKA activation, identification of cAMP-dependent phosphorylation sites is key. Phosphoproteomics can help link this cAMPmediated PKA regulation to downstream effector molecules and biological functions (Beltejar et al, 2017). Mass spectrometry can identify thousands of phosphosites with high precision (Altelaar et al, 2013) and quantitative mass spectrometry has been shown to be sensitive to dynamic changes in protein phosphorylation following GPCR activation (Williams et al, 2016).…”