Cardiac-type sarco(endo)plasmic reticulum Ca(2)-ATPase (SERCA2a) plays a major 
role in cardiac muscle contractility. Phospholamban (PLN) regulates the function 
of SERCA2a via its Ser(16)-phosphorylation. Since it has been proposed that the 
Ser/Thr residues on cytoplasmic and nuclear proteins are modified by O-linked 
N-acetylglucosamine (O-GlcNAc), we examined the effect of O-GlcNAcylation on PLN 
function in rat adult cardiomyocytes. Studies using enzymatic labeling and 
co-immunoprecipitation of wild type and a series of mutants of PLN showed that 
PLN was O-GlcNAcylated and Ser(16) of PLN might be the site for O-GlcNAcylation. 
In cardiomyocytes treated with 
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), 
the O-GlcNAcylation was significantly increased compared to non-treated cells. 
Simultaneously, Ser(16)-phosphorylation of PLN was reduced. In Chinese hamster 
ovary cells where PLN cDNA and O-GlcNAc transferase siRNA were co-transfected, 
the Ser(16)-phosphorylation of PLN was significantly increased compared to 
controls. The same results were observed in heart homogenates from diabetic 
rats. In a co-immunoprecipitation of PLN with SERCA2a, the physical interaction 
between the two proteins was increased in PUGNAc-treated cardiomyocytes. Unlike 
non-treated cells, the activity of SERCA2a and the profiles of calcium 
transients in PUGNAc-treated cardiomyocytes were not significantly changed even 
after treatment with catecholamine. These data suggest that PLN is 
O-GlcNAcylated to induce the inhibition of its phosphorylation, which correlates 
to the deterioration of cardiac function. This might define a novel mechanism by 
which PLN regulation of SERCA2a is altered under conditions where 
O-GlcNAcylation is increased, such as those occurring in diabetes.