Lethal, Hereditary Mutants of Phospholamban Elude Phosphorylation by Protein Kinase A*

Background: Heterozygous mutations in the cytoplasmic domain of phospholamban cause lethal dilated cardiomyopathy. Results: The mutations alter phospholamban-protein kinase A interactions that are essential for substrate recognition and phosphorylation. Conclusion: Hereditary mutations in phospholamban that prevent phosphorylation by protein kinase A will lead to chronic inhibition of SERCA. Significance: Arginines in the cytoplasmic domain of phospholamban should be considered hot spots for hereditary mutations leading to dilated cardiomyopathy. The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg9 to Cys, Arg9 to Leu, Arg9 to His, and Arg14 deletion, alter regulation of SERCA. Deletion of Arg14 disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg9 causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg9 in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.