Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Abstract D. M. Valks, S. A. Cook, F. H. Pham, P. R. Morrision, A. Clerk and P. H. Sugden. Phenylephrine Promotes Phosphorylation of Bad in Cardiac Myocytes Through the Extracellular Signal-regulated Kinases 1/2 and Protein Kinase A. Journal of Molecular and Cellular Cardiology (2002) 34 , 749–763. Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 μ M ) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 μ M ), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the α-adrenoceptor, but, at high concentrations (100 μ M ), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the β-adrenoceptor. Phenylephrine (100 μ M ) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both α 1 - and β-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the β-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.