Regulation of cAMP-dependent protein kinase subunit expression in CATH.a and SH-SY5Y cells.
Increasing evidence supports a role for adaptations in the cAMP pathway in mediating aspects of neural plasticity. These adaptations include altered levels of the catalytic (C) and regulatory (R) subunits of cAMP-dependent protein kinase (PKA) in specific neuronal cell types. In an effort to understand the mechanisms underlying this regulation of PKA, the effects of perturbing the cAMP pathway on PKA expression were examined in the locus ceruleus-like CATH.a cell line and the human neuroblastoma SH-SY5Y cell line. Exposure of CATH.a and SH-SY5Y cells to forskolin, a direct activator of adenylyl cyclase, resulted in a time-dependent decrease in levels of immunoreactivity of C and the two types of R (RI and RII). This decrease in PKA subunit immunoreactivity was not attenuated by pretreatment of the cells with the protein synthesis inhibitor cycloheximide. Moreover, exposure of the cell lines to forskolin had no effect on levels of mRNA for these PKA subunits over a wide time course. In contrast, treatment of cells with a cAMP antagonist (Rp-8-bromo-cAMPS) dramatically increased levels of PKA subunit immunoreactivity, particularly that of RI. No change in RI mRNA levels, however, was observed under these conditions. The PKA catalytic inhibitor H-89 did not attenuate the forskolin-induced down-regulation. The PKA subunit down-regulation was blocked, however, by treatment of the cells with Leu-Leu-Leu or lactacystin, inhibitors of proteasomes that are implicated in the regulated proteolysis of specific cellular proteins. Together, these findings demonstrate that regulation of PKA subunit expression by forskolin or a cAMP antagonist occurs primarily through post-transcriptional mechanisms and suggests the involvement of proteasome-mediated degradation in these phenomena.
Division of Molecular Psychiatry, Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06508, USA.
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Boundy
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Chen
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Nestler
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009097:Multienzyme Complexes / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D009097:Multienzyme Complexes; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex