Melatonin stimulates calmodulin phosphorylation by protein kinase C.
Calmodulin (CaM)-dependent processes can be modulated by the availability of Ca(+2), the subcellular distribution of both CaM and its target proteins, CaM antagonism, and post-translational modifications such as CaM phosphorylation. Melatonin, the pineal secretory product synthesized during the dark phase of the photoperiod is an endogenous CaM antagonist. This indolamine causes CaM subcellular redistribution in epithelial MDCK and MCF-7 cells, and selectively activates protein kinase C alpha (PKC alpha) in neuronal N1E-115 cells. In the present work we have characterized the phosphorylation of CaM mediated by PKC alpha and its stimulation by melatonin in an in vitro reconstituted enzyme system. Additionally, the participation of MAPK and ERKs, downstream kinases of the PKC signaling pathway, was explored utilizing MDCK cell extracts as source of these kinases. Phosphorylation of CaM was characterized in the whole cells by MDCK cell metabolic labeling with [(32)P]-orthoposhospate, and CaM separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, as well as by immunocolocalization of phosphorylated threonine/serine residues and CaM in cultured cells incubated with melatonin. Our results show that melatonin increased CaM phosphorylation by PKC alpha with an EC(50) of 10(-8) m in the presence of the phorbol ester, phorbol-12-myristate-13-acetate (PMA) in the in vitro reconstituted enzyme system. An increase in phosphorylated CaM was also observed in cells cultured with melatonin, or PMA for 2 hr, while, PKC, MAPK, or ERK inhibitors abolished CaM phosphorylation elicited by melatonin in MDCK cell extracts. Our data show that melatonin can stimulate phosphorylation of CaM by PKC alpha in the in vitro reconstituted system and suggest that in MDCK cells this phosphorylation is accomplished by PKC. Modification of CaM by melatonin can be another route to inhibit CaM interaction with its target enzymes.
Depto. Neurofarmacología, Subdirección de Investigaciones Clínicas, Instituto Nacional de Psiquiatría, México.
Elena
E
Soto-Vega
Isaura
I
Meza
Gerardo
G
Ramírez-Rodríguez
Gloria
G
Benitez-King
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002457:Cell Extracts; D045744:Cell Line, Tumor; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008550:Melatonin / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology
D000975:Antioxidants; D002147:Calmodulin; D002457:Cell Extracts; D004791:Enzyme Inhibitors; C016658:phorbolol myristate acetate; D011493:Protein Kinase C; D020929:Mitogen-Activated Protein Kinase Kinases; D008550:Melatonin; D013755:Tetradecanoylphorbol Acetate