Endothelins stimulate tyrosine phosphorylation and activity of p42/mitogen-activated protein kinase in astrocytes.
Endothelins (ET-1, -2, -3) display pleiotropic activities, by signalling through G-protein-coupled membrane receptors. We show here that ET-1 and ET-3 stimulate within minutes the tyrosine phosphorylation of a 42 kDa protein (p42) in primary cultures of mouse embryo astrocytes, but not in any of two subclones of rat astrocytoma C6 cells. This effect, measured by anti-phosphotyrosine immunoblotting of cell extracts, was also observed in response to bradykinin, platelet-derived growth factor, the phorbol ester phorbol 12-myristate 13-acetate and the G-protein activator fluoroaluminate. Pretreatment of cells with pertussis toxin, which inactivates Gi/G(o) proteins, did not affect these responses. However, down-regulation of protein kinase C completely blocked the response to phorbol ester and fluoroaluminate and at least partially impaired the ET-1-stimulated phosphorylation of p42. We have identified p42 as p42mapk, a mitogen-activated protein (MAP) kinase, on the basis of the following data: by sequential immunoblotting with antiphosphotyrosine and anti-MAP kinase antibodies, (i) similar kinetics are observed for p42 phosphorylation and the decrease in p42mapk electrophoretic mobility, likely corresponding to its tyrosine/threonine phosphorylation [de Vries-Smits, Boudewijn, Burgering, Leevers, Marshall and Bos (1992) Nature (London) 357, 602-604]; (ii) p42 and the shifted form of p42mapk co-migrate on SDS/PAGE; (iii) the myelin-basic-protein kinase activity of p42mapk is stimulated by ET-1, in parallel with the tyrosine phosphorylation of p42. In conclusion, these findings strongly suggest that endothelins can stimulate the tyrosine phosphorylation and activation of p42mapk in astrocytes, via pertussis-toxin-insensitive G protein and protein kinase C-dependent and -independent pathways.
Cazaubon
S
S
Laboratoire d'Immuno-Pharmacologie Moléculaire, Institut Cochin de Génétique Moléculaire, CNRS UPR, Université Paris VII, France.
Parker
P J
PJ
Strosberg
A D
AD
Couraud
P O
PO
D000535:Aluminum / Q000494:pharmacology; D017607:Aluminum Compounds; D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D001254:Astrocytoma; D002999:Clone Cells; D016232:Endothelins / Q000494:pharmacology*; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D008024:Ligands; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D017607:Aluminum Compounds; D016232:Endothelins; D008024:Ligands; D014443:Tyrosine; D000535:Aluminum; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D038362:Glycogen Synthase Kinase 3; D013755:Tetradecanoylphorbol Acetate; D005459:Fluorides; C032311:aluminum fluoride