Distinct Functional Effects of Phospholamban Phosphorylation States

Abstract

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). It is known that PLB inhibits SERCA and that PLB phosphorylation at S16 and/or T17 relieves this inhibition in cardiac SR. However, the functional effects of the two PLB phosphorylation sites have not been compared quantitatively. Therefore, we co-reconstituted SERCA1a with synthetic PLB, pS16-PLB, pT17-PLB, or pS16/pT17-PLB in phospholipid vesicles and measured Ca2+-dependent SERCA activity. Inhibition was maximally relieved by phosphorylation at S16, followed by doubly phosphorylated PLB (pS16/pT17-PLB) and pT17-PLB. Thus, T17 phosphorylation shows the least capacity for SERCA inhibition relief, and actually decreases the effect of S16 phosphorylation on the same PLB. Quantitative Western blots were used to demonstrate that all four PLB phosphorylation states are present in cardiac SR. Cardiac SR was treated with PLB antibodies and protein kinase A (PKA) to quantify the effects of PLB phosphorylation states on SERCA inhibition in a biological milieu. These results show that each PLB phosphorylation state uniquely alters Ca2+ homeostasis. These effects are relevant to cardiovascular health, disease and treatment.View Large Image | View Hi-Res Image | Download PowerPoint Slide