Endothelin Stimulates Mitogen-Activated Protein Kinase p42 Activity Through the Phosphorylation of the Kinase in Rat Mesangial Cells

Abstract

Summary: We have reported that endothelin-1 (ET-1), which is a constrictor and mitogenic peptide, can increase mitogen-activated protein kinase p42 (p42^mapk) activity in rat mesangial cells. In this study, we investigate the mechanism of activation of p42^mapk. Treatment of quiescent mesangial cells with 10^-7 M ET-1 biphasically stimulated p42^mapk activity. The kinetics of the immunoprecipitated p42^mapk activity induced by ET-1 showed a maximal 3.5-to 4.5-fold stimulation 5 min after the addition of the agonist to the cell cultures and a smaller, 2.5-fold increase of activity between 2 and 6 h after ET challenge. Neither peak of p42^mapk activity induced by ET-1 was inhibited by pretreatment of the cells with either cycloheximide to inhibit protein synthesis or actinomycin D to retard transcription. Analysis by immunoblot showed that p42^mapk was not affected by these pretreatments. In addition, the kinetics of phosphorylation of p42^mapk showed a significant ³²p incorporation into p42 at 5, 30, and 240 min after ET stimulation. Because phosphorylation on tyrosine and threonine residues of the enzyme is necessary for activation of the kinase, we believe that the phosphorylation of the p42^mapk rather than transcriptional or translational induction is responsible for the activation of p42^mapk in mesangial cells stimulated with ET-1.