Phenylephrine Promotes Phosphorylation of Bad in Cardiac Myocytes Through the Extracellular Signal-regulated Kinases 1/2 and Protein Kinase A

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 μM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 μM), 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 μM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the β-adrenoceptor. Phenylephrine (100 μM) 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 α1- 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.