Phosphorylation of phospholamban at threonine-17 in the absence and presence of beta-adrenergic stimulation in neonatal rat cardiomyocytes.
The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5+/-6.3% and 92. 5+/-11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1+/-0.3% and 8.6+/-2. 1%, respectively. However, the effect of KN-93 was attenuated (47. 8+/-3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.
Bartel
S
S
Max Delbrück Center for Molecular Medicine, Berlin, 13125, Germany. molcard@mdc-berlin.de
Vetter
D
D
Schlegel
W P
WP
Wallukat
G
G
Krause
E G
EG
Karczewski
P
P
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D013997:Time Factors; D000099211:Phospholamban
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D000318:Adrenergic beta-Agonists; D002118:Calcium; D002120:Calcium Channel Agonists; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000242:Cyclic AMP; D004791:Enzyme Inhibitors; D007476:Ionophores; D007545:Isoproterenol; D019319:Okadaic Acid; D012433:Ryanodine; D012694:Serine; D019284:Thapsigargin; D013912:Threonine; D000099211:Phospholamban