Phospholamban degradation is induced by phosphorylation-mediated ubiquitination and inhibited by interaction with cardiac type Sarco(endo)plasmic reticulum Ca2+-ATPase

Abstract

Phospholamban (PLN) regulates cardiac type sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA2a) via Ser16-phosphorylation. During heart failure, PLN expression is downregulated with SERCA2a; however, the mechanism of its regulation is not fully understood. Phosphorylation triggers protein degradation and because PLN phosphorylation is upregulated in failing hearts, we examined whether PLN is degraded by Ser16-phosphorylation. Cells overexpressing PLN exhibited its degradation post isoproterenol (Iso), forskolin, or 3-isobutyl-1-methylxanthine (IBMX) addition. Moreover, this degradation was inhibited by a cAMP-dependent protein kinase (PKA) inhibitor––H89. Co-immunoprecipitation revealed that Lys3 of PLN was oligo-ubiquitinated when ubiquitin was overexpressed, and was degraded by Iso treatment. However, when co-expressed with SERCA2a, oligo-ubiquitinated PLN at Lys3 was not degraded by Iso treatment. In failing hearts from 16 week-old TgPLNR9C mice, oligo-ubiquitinated PLN levels increased and PLN expression was downregulated. Furthermore, SERCA2a mRNA levels in TgPLNR9C mice hearts were lower than that in wild type mice; however, PLN mRNA levels showed no changes. In another heart failure model, MG132 treatment reversed PLN degradation. These data suggest that PLN is, at least partially, oligo-ubiquitinated at Lys3 and degraded through Ser16-phosphorylation-mediated poly-ubiquitination during heart failure.