The sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) is critical for 
sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR) and 
regulating cardiac muscle relaxation. Protein-protein interactions indicated 
that it exists in complex with Ca(2+)/calmodulin-dependent protein kinase II 
(CaMKII) and its anchoring protein alphaKAP. Confocal imaging of isolated 
cardiomyocytes revealed the colocalization of CAMKII and alphaKAP with SERCA2a 
at the SR. Deletion analysis indicated that SERCA2a and CaMKII bind to different 
regions in the association domain of alphaKAP but not with each other. Although 
deletion of the putative N-terminal hydrophobic amino acid stretch in alphaKAP 
prevented its membrane targeting, it did not influence binding to SERCA2a or 
CaMKII. Both CaMKIIdelta(C) and the novel CaMKIIbeta(4) isoforms were found to 
exist in complex with alphaKAP and SERCA2a at the SR and were able to 
phosphorylate Thr-17 on phospholamban (PLN), an accessory subunit and known 
regulator of SERCA2a activity. Interestingly, the presence of alphaKAP was also 
found to significantly modulate the Ca(2+)/calmodulin-dependent phosphorylation 
of Thr-17 on PLN. These data demonstrate that alphaKAP exhibits a novel 
interaction with SERCA2a and may serve to spatially position CaMKII isoforms at 
the SR and to uniquely modulate the phosphorylation of PLN.