摘要
We now know that 11 different families of cyclic nucleotide phosphodiesterases(PDEs) are expressed in mammalian species. Most of these families contain multiple gene products and most of the genes utilize alternative splicing or alternative start sites to encode more than one RNA/protein.However,specific functions for these different PDEs have not yet been identified in most cell types despite the fact that selective inhibitors to most of the PDE families are available. Conventional approaches to study PDE function typically rely on measurements of global cAMP,or general increases in cAMP-dependent protein kinase A(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 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 that are regulated by specific cAMP-PDEs(the PDE-regulated phosphoproteomes). In MA-10 Leydig cells we find that in order to detect appreciable increased in either phosphorylation or steroid production,one needs to inhibit both PDE 4 and PDE 8 activity.Using this combination of inhibitors,we find large PDE inhibitor-induced changes in many different proteins that modulate steroid trafficking and biosynthesis. The data are consistent with the idea that cAMP serves to coordinate hormone stimulation of steroid production by altering the phosphorylation of many different proteins at multiple points in the overall pathway rather than just controlling a single rate limiting step. It seems quite likely that many of the proteins phosphorylated in this cell type in response to PDE inhibition,will also be regulated by cAMP in many other cell types.Similarly,in Jurkat cells we find multiple,distinct,PDE regulated phosphoproteomes that differ in response to different PDE inhibitors. Here we also find that little phosphorylation occurs unless at least 2 different PDEs are concurrently inhibited in these cells. Inhibition of a single PDE produces little effect. Bioinformatics analyses of these phosphoproteomes suggest differing functional roles,mechanisms of action,and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. In this tissue also,the data strongly imply that phosphorylation of many different substrates contribute to cAMP-dependent regulation of these cells. Overall,the findings illustrate 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.
We now know that 11 different families of cyclic nucleotide phosphodiesterases(PDEs) are expressed in mammalian species. Most of these families contain multiple gene products and most of the genes utilize alternative splicing or alternative start sites to encode more than one RNA/protein.However,specific functions for these different PDEs have not yet been identified in most cell types despite the fact that selective inhibitors to most of the PDE families are available. Conventional approaches to study PDE function typically rely on measurements of global cAMP,or general increases in cAMP-dependent protein kinase A(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 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 that are regulated by specific cAMP-PDEs(the PDE-regulated phosphoproteomes). In MA-10 Leydig cells we find that in order to detect appreciable increased in either phosphorylation or steroid production,one needs to inhibit both PDE 4 and PDE 8 activity.Using this combination of inhibitors,we find large PDE inhibitor-induced changes in many different proteins that modulate steroid trafficking and biosynthesis. The data are consistent with the idea that cAMP serves to coordinate hormone stimulation of steroid production by altering the phosphorylation of many different proteins at multiple points in the overall pathway rather than just controlling a single rate limiting step. It seems quite likely that many of the proteins phosphorylated in this cell type in response to PDE inhibition,will also be regulated by cAMP in many other cell types.Similarly,in Jurkat cells we find multiple,distinct,PDE regulated phosphoproteomes that differ in response to different PDE inhibitors. Here we also find that little phosphorylation occurs unless at least 2 different PDEs are concurrently inhibited in these cells. Inhibition of a single PDE produces little effect. Bioinformatics analyses of these phosphoproteomes suggest differing functional roles,mechanisms of action,and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. In this tissue also,the data strongly imply that phosphorylation of many different substrates contribute to cAMP-dependent regulation of these cells. Overall,the findings illustrate 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.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2017年第5期445-446,共2页
Chinese Journal of Pharmacology and Toxicology
