title: Phosphorylation and down-regulation of Bim by muscarinic cholinergic receptor activation via protein kinase C.
abstract: Bim is one of the proapoptotic BH3-only homologs of the Bcl-2 family proteins, which interacts with other Bcl-2 family proteins to activate the intrinsic apoptotic pathway. The expression and protein level of Bim are highly regulated in cells at both transcriptional and post-translational levels, and inadequate control of Bim level may largely determine its pro-apoptic activity. In the present study, we reported that carbachol, a muscarinic cholinergic receptor agonist, regulated Bim in human SH-SY5Y neuroblastoma cells. Carbachol rapidly induced an upward gel mobility shift of Bim, which was abolished by protein phosphatase treatment, indicating an increased Bim phosphorylation by carbachol. The effect of carbachol was mimicked by the protein kinase C activator 12-myristate 13-acetate (PMA) and was blocked by the protein kinase C inhibitor rottlerin, suggesting that activation of protein kinase C was required for carbachol-induced phosphorylation of Bim. Prolonged treatment with carbachol and PMA significantly decreased Bim protein levels in total cell lysates and mitrochondria. Carbachol and PMA had no effect in the transcriptional regulation of Bim, whereas the reduction of Bim by both carbachol and PMA was reversed by the proteosome inhibitors, suggesting that carbachol and PMA facilitated the proteosome-dependent Bim degradation. Thus, this study identified the muscarinic receptor-protein kinase C signaling pathway as a regulator of Bim in neuroblastoma cells, and activation of muscarinic receptor and protein kinase C functions to induce Bim phosphorylation, followed by down-regulation of the proapoptotic protein.
authors.affiliation: Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Styles
authors.forename: Wawa
authors.initials: W
authors.lastname: Zhu
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Li
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D001578:Benzopyrans / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000098:Acetophenones; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D018721:Muscarinic Agonists; D061988:Proteasome Inhibitors; D011518:Proto-Oncogene Proteins; D011976:Receptors, Muscarinic; D002217:Carbachol; C085746:rottlerin; D011493:Protein Kinase C; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate