Abstract
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.
Highlights
The data here show that ␣KAP can exist in a complex with calmodulin-dependent protein kinase II (CaMKII) isoforms, sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a), and CaM at the cardiac sarco-endoplasmic reticulum (SR). ␣KAP directly binds SERCA2a as well as recruits CaMKII␦C and CaMKII4 isoforms, which are prominent in cardiac tissue
SERCA2a binds directly to regions in the association domain of ␣KAP that are distinct from those that associate with CaMKII
The removal of the putative transmembrane domain of ␣KAP disengaged it from the SR membrane, as reported [15, 30], it did not effect its interaction with SERCA2a or CAMKII, since the association domain of ␣KAP containing the binding sites is extramembranous
Summary
Expression Constructs of CaMKII␦C, CaMKIIC, ␣KAP, SERCA2a, and Phospholamban—The cloning of CaMKII4 and ␣KAP from myocardium has been described previously [14]. Immunofluorescence—Cells transfected with different cDNA expression constructs were fixed with 4% paraformaldehyde for 10 min and washed four times with phosphate-buffered saline. Phosphorylation Assays—Phosphorylation of SERCA2a was carried out by co-transfecting SERCA2a-GFP and ␣KAP-Myc with either CaMKII␦C-Myc or CaMKII4-Myc. IP reactions were performed with Myc-agarose as described above, and precipitated proteins were incubated in assay buffer (20 mM MOPS, pH 7.2, 25 mM -glycerol phosphate, 1 mM sodium orthovandate, 1 mM dithiothreitol, either 1 mM CaCl2 plus 100 nM calmodulin or 5 mM EGTA, and [␥-32P]ATP (0.4 ϫ 10Ϫ5 cpm)) for 2 min at room temperature; the reaction was terminated with 50 l of SDS gel loading buffer; proteins were resolved on SDS-PAGE and gel-dried; and phosphoproteins were detected on x-ray films. Proteins were resolved on SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and immunoblotted with antiCaMKII polyclonal antibody or phospho-PLN antibody (Badrilla, UK) or anti-PLN antibody (Affinity Bioreagent)
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