Purified, Reconstituted Cardiac Ca2+-ATPase Is Regulated by Phospholamban but Not by Direct Phosphorylation with Ca2+/Calmodulin-dependent Protein Kinase

Abstract

Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to ␤-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca 2؉ -ATPase). Recently, direct phosphorylation of Ca 2؉ -ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca 2؉ -ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (ϳ1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by ϳ50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca -ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on V max for calcium transport.

Functional Co-expression of the Canine Cardiac Ca2+Pump and Phospholamban in Spodoptera frugiperda (Sf21) Cells Reveals New Insights on ATPase Regulation

“…Thus, use of the baculovirus cell expression system provides additional strong evidence that the main regulatory effect of phospholamban is on the apparent Ca 2ϩ affinity of the Ca 2ϩ pump, but not on the V max of the enzyme. A similar regulatory effect of phospholamban on the Ca 2ϩ pump has been reported with use of canine cardiac sarcoplasmic reticulum vesicles (4,6,23,42), with use of the purified and reconstituted proteins (16,20), with use of the recombinant proteins expressed in HEK-293 membranes (43), and with use of phospholamban knockout mice (10). Although Kirchberger and co-workers (44) have recently challenged the idea that the main regulatory effect of phospholamban is on the K Ca value for Ca 2ϩ transport, most evidence concurs that dephosphorylated phospholamban strongly suppresses the Ca 2ϩ affinity of the enzyme, with an insignificant effect on V max .…”

“…The purified, recombinant protein was successfully reconstituted into proteoliposomes to study its secondary structure (18) and oligomeric organization in the lipid bilayer (19). Successful co-reconstitution with Ca 2ϩ pumps purified from rabbit skeletal muscle (16) and canine myocardium (20) was also achieved. Here, we report on the further development of this system for functional co-expression of phospholamban with the canine cardiac Ca 2ϩ pump (SERCA2a).…”

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

“…The phosphorylation of SERCA2 on Ser-38 has been described as a regulatory feature capable of very significant activation of Ca 2ϩ -ATPase activity (24,26), although its occurrence and implication have been disputed (14,30). This site, although unique to SERCA2, is contained within a segment of the protein which is highly conserved between SERCA1 and SERCA2 (Fig.…”

“…These Ca 2ϩ -free conditions were similar to the "control" conditions of Xu et al (24), making it unclear whether Xu et al (24) had described stimulation of pump activity in response to CaMKII phosphorylation or artifactual inhibition of pump activity under (Ca 2ϩ -free) control conditions. In separate studies Ready et al (14) explored the phosphorylation of SERCA using highly purified cardiac SR and highly purified SERCA2a. CaMKII was unable to phosphorylate SERCA in either of these preparations, however the phosphorylation of a junctional SR protein of 100 kDa was noted.…”

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

“…Some reports attributed enhanced SERCA V max to CaMKII dependent SERCA phosphorylation [43,44]. However, such a direct CaMKIIdependent SERCA phosphorylation has been strongly challenged by others [45][46][47].…”