CONCERTED REGULATION OF CARDIAC SARCOPLASMIC-RETICULUM CALCIUM-TRANSPORT BY CYCLIC ADENOSINE-MONOPHOSPHATE DEPENDENT AND CALCIUM-CALMODULIN-DEPENDENT PHOSPHORYLATIONS

The rate of calcium uptake by canine cardiac sarcoplasmic reticulum is regulated by both a cyclic adenosine monophosphate (cAMP) dependent phosphorylation and a Ca2+-calmodulin-dependent phosphorylation. The pure catalytic subunit of cAMP-dependent protein kinase catalyzes directly the incorporation of phosphate into phospholamban. This phosphorylation is not Ca2+-dependent. In the presence of Ca2+ and calmodulin, a membrane-bound protein kinase catalyzes the incorporation of the same amount of phosphate into phospholamban at a different site. The latter covalent modification is totally dependent on Ca2+ and on exogenous calmodulin, with the half-maximal initial rate at 70 nM calmodulin. Calmodulin binding sites are shown to be present on sarcoplasmic reticulum vesicles by using [125I]calmodulin. The membrane-bound calmodulin-dependent kinase is not inhibited by the cAMP-dependent protein kinase inhibitor. It does phosphorylate exogenous glycogen phosphorylase b. Conversely, exogenous glycogen phosphorylase b kinase catalyzes phospholamban phosphorylation beyond the activation provided by the attached δ subunit (calmodulin) alone. When fully phosphorylated, phospholamban is dephosphorylated by phosphoprotein phosphatase with biphasic kinetics. Whereas the Ca2+-Mg2+-ATPase activity is not modified by the covalent modifications of phospholamban, the rate of Ca2+ uptake is increased under conditions which induce phospholamban phosphorylation by the calmodulin-dependent kinase. This effect is amplified by the cAMP-dependent phosphorylation. A hypothetical model is presented for the functional role of these phosphorylations according to which each ATPase monomer interacts with one phospholamban monomer of M, 11 000. The short stretch of phospholamban exposed at the outer surface of the vesicles contains both sites of phosphorylation, perhaps in a double-headed configuration. The membrane-bound kinase, similar but not identical to phosphorylase kinase, acts as a sensor of the cytosolic free Ca2+ concentration, whereas the cAMP-dependent direct phosphorylation provides an explanation for the acceleration of relaxation induced by catecholamines. There is no evidence for a metabolic cascade that would involve sequentially cAMP-dependent and calmodulin-dependent protein kinases. © 1979, American Chemical Society. All rights reserved.