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 exchange protein activated by cAMP (EPAC) activity. Although newer approaches utilizing subcellularly-targeted FRET reporter sensors have helped to 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 used 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 found multiple, distinct phosphoproteomes that differ in response to different PDE inhibitors. We also found that little phosphorylation occurs unless at least 2 different PDEs are concurrently inhibited in these cells. Moreover, bioinformatics analyses of these phosphoproteomes provides insight into the 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 phosphorylation of many different substrates contribute to cAMP-dependent regulation of these cells. The findings further indicate 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.