Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum
Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum
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DOI：
10.1074/jbc.m109.031989
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发表时间：
2009-11-06
影响因子：
4.8
通讯作者：
Kim, Do Han
中科院分区：
文献类型：
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作者：
Sahoo, Sanjaya Kumar;Kim, Taeyong;Kim, Do Han
Calumenin是一种位于肌浆网（SR）中的多EF-手型Ca 2+结合蛋白，具有C-末端SR滞留信号HDEF。最近，我们显示了钙调素在大鼠心脏SR中与SERCA 2相互作用的证据（Sahoo，S. K.，和Kim，D. H.（2008）Mol.细胞26，265-269）。本研究通过各种基因操作方法进一步表征小鼠心脏中calumenin与SERCA 2的关联。免疫细胞化学分析显示calumenin和SERCA 2部分共定位于HL-1细胞中。在HL-1细胞中进行calumenin的敲低（KD），并且80%的calumenin的敲低不引起其它Ca 2+循环蛋白的任何表达变化。但它增强了Ca 2+瞬时振幅，并显示达到峰值的时间缩短，达到基线50%的时间缩短。草酸盐支持的Ca 2+摄取显示钙调素KD HL-1细胞中SERCA 2的Ca 2+敏感性增加。Calumenin和SERCA 2的相互作用显着降低毒胡萝卜素，钒酸盐，或ATP的存在下，相比1.3 μ M的Ca 2+，这表明该相互作用是有利于在E1状态的SERCA 2。钙调素缺失片段和SERCA 2管腔结构域的谷胱甘肽S-转移酶下拉测定表明钙调素的132-222个氨基酸和SERCA 2-L4的853-892个氨基酸的区域是主要的结合伴侣。根据我们的体外结合数据和现有的信息的三维结构的Ca 2 +-ATP酶，calumenin和SERCA 2之间的相互作用的分子模型提出。以上结果提示calumenin是一种新的SERCA 2调节因子，其表达变化与心肌细胞Ca ~（2+）循环密切相关。
Calumenin is a multiple EF-hand Ca2+-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 Ca2+-cycling proteins. But it enhanced Ca2+ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca2+ uptake showed increased Ca2+ 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 mu M Ca2+, 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 Ca2+-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 Ca2+-cycling of cardiomyocytes.