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Endothelin Induces a Calcium‐Dependent Phosphorylation of PEA‐15 in Intact Astrocytes: Identification of Ser 104 and Ser 116 Phosphorylated, Respectively, by Protein Kinase C and Calcium/Calmodulin Kinase II In Vitro
Author(s) -
Kubes Miroslav,
Cordier Jocelyne,
Glowinski Jacques,
Girault JeanAntoine,
Chneiweiss Hervé
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.71031307.x
Subject(s) - protein kinase a , phosphorylation , protein kinase c , map2k7 , mitogen activated protein kinase kinase , protein phosphorylation , biology , casein kinase 2 , cyclin dependent kinase 2 , biochemistry , kinase , cgmp dependent protein kinase , map kinase kinase kinase , casein kinase 1 , calmodulin , microbiology and biotechnology , enzyme
PEA‐15 (phosphoprotein enriched in astrocytes, M r = 15,000) is an acidic serine‐phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine‐induced apoptosis. PEA‐15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA‐15 phosphorylation. Endothelin‐1 or endothelin‐3 (0.1 µ M ) induced a robust phosphorylation of PEA‐15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA‐15 following endothelin‐1 treatment identified two phosphorylated residues: Ser 104 , previously recognized as the protein kinase C site, and a novel phosphoserine, Ser 116 , located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin‐dependent protein kinase II (CaMKII). Partly purified PEA‐15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two‐dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser 116 and had no effect on Ser 104 , but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA‐15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.