Immunodetection of phosphorylation sites gives new insights into the mechanisms underlying phospholamban phosphorylation in the intact heart.
Phosphorylation site-specific antibodies, quantification of 32P incorporation into phospholamban, and simultaneous measurements of mechanical activity were used in Langendorff-perfused rat hearts to provide further insights into the underlying mechanisms of phospholamban phosphorylation. Immunological detection of phospholamban phosphorylation sites showed that the isoproterenol concentration-dependent increase in phospholamban phosphorylation was due to increases in phosphorylation of both Ser16 and Thr17 residues. When isoproterenol concentration was increased at extremely low Ca2+ supply to the myocardium, phosphorylation of Thr17 was virtually absent. Under these conditions, 32P incorporation into phospholamban, due to Ser16, decreased by 50%. Changes in Ca2+ supply to the myocardium either at constant beta-adrenergic stimulation or in the presence of okadaic acid, a phosphatase inhibitor, exclusively modified Thr17 phosphorylation. Changes in phospholamban phosphorylation due to either Ser16 and/or Thr17 were paralleled by changes in myocardial relaxation. The results indicate that cAMP- (Ser16) and Ca2+-calmodulin (Thr17)-dependent pathways of phospholamban phosphorylation can occur independently of each other. However, in the absence of beta-adrenergic stimulation, phosphorylation of Thr17 could only be detected after simultaneous activation of Ca2+-calmodulin-dependent protein kinase and inactivation of phosphatase. It is suggested that under physiological conditions, this requisite is only filled by cAMP-dependent mechanisms.
Mundiña-Weilenmann
C
C
Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, 1900 La Plata, Argentina. cicme@isis.unlp.edu.ar
Vittone
L
L
Ortale
M
M
de Cingolani
G C
GC
Mattiazzi
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007545:Isoproterenol; D019319:Okadaic Acid; D012694:Serine; D013912:Threonine; D000099211:Phospholamban