期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：30
页码：E6240-E6249
DOI：10.1073/pnas.1703939114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
