Synthetic Phosphopeptides Enable Quantitation of the Content and Function of Phospholamban’s four Phosphorylation States in Cardiac Muscle [Membrane Biology]

September 4th, 2014 by Ablorh, N.-A. D., Dong, X., James, Z. M., Xiong, Q., Zhang, J., Thomas, D. D., Karim, C. B.

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA). It has been shown that unphosphorylated PLB (U-PLB) inhibits SERCA and that phosphorylation of PLB at S16 or T17 relieves this inhibition in cardiac SR. However, the levels of the four phosphorylation states of PLB (U-PLB, P16-PLB, P17-PLB, and doubly phosphorylated 2P-PLB) have not been measured quantitatively in cardiac tissue, nor have their functional effects on SERCA been determined directly. We have solved both problems through the chemical synthesis of all four PLB species. We first used the synthetic PLB as standards for a quantitative immunoblot assay, to determine the concentrations of all four PLB phosphorylation states in pig cardiac tissue, with and without left ventricular hypertrophy (LVH) induced by aortic-banding. In both LVH and sham hearts, all phosphorylation states were significantly populated, but LVH hearts showed a significant decrease in U-PLB, with a corresponding increase in the ratio of total phosphorylated-PLB to U-PLB. To determine directly the functional effects of each PLB species, we co-reconstituted each of the synthetic peptides in phospholipid membranes with SERCA and measured Ca-dependent ATPase activity. SERCA inhibition was maximally relieved by P16-PLB (the most highly populated PLB state in cardiac SR), followed by 2P-PLB, then P17-PLB. These results show that each PLB phosphorylation state uniquely alters Ca2+ homeostasis, with important implications for cardiac health, disease and therapy.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Synthetic Phosphopeptides Enable Quantitation of the Content and Function of Phospholamban’s four Phosphorylation States in Cardiac Muscle [Membrane Biology]