Characterization of calumenin-SERCA2 interaction in mouse cardiac sarcoplasmic reticulum.
Calumenin is a multiple EF-hand Ca(2+)-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265-269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca(2+)-cycling proteins. But it enhanced Ca(2+) transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca(2+) uptake showed increased Ca(2+) sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 mum Ca(2+), suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132-222 amino acids of calumenin and 853-892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca(2+)-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca(2+)-cycling of cardiomyocytes.
Sahoo
Sanjaya Kumar
SK
Department of Life Science and Systems Biology Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Kim
Taeyong
T
Kang
Gil Bu
GB
Lee
Jung-Gyu
JG
Eom
Soo Hyun
SH
Kim
Do Han
DH
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D051379:Mice; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D002135:Calcium-Binding Proteins; C107182:Calu protein, mouse; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506947:Atp2a2 protein, mouse