A novel particulate form of Ca2+/CaMKII-dependent protein kinase II in neurons
A novel particulate form of Ca2+/CaMKII-dependent protein kinase II in neurons
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DOI：
10.1523/jneurosci.20-09-03076.2000
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发表时间：
2000-05-01
影响因子：
5.3
通讯作者：
Cheng, JHT
中科院分区：
文献类型：
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作者：
Dosemeci, A;Reese, TS;Cheng, JHT
来自前脑的细胞骨架和突触后密度(PSD)组分含有离散的球形结构，这些结构对钙/钙调蛋白依赖的蛋白激酶II(CaMKII)免疫阳性。旋转阴影电子显微镜观察到的球形结构的平均直径类似于100 nm，与突触后密度不同，不标记PSD-95。通过去污剂N-十二烷基肌氨酸酯的提取，这些结构被纯化到接近均一的水平。生化分析表明，在纯化的制剂中，CaMKII几乎占了所有的蛋白质，表明球形结构是自结合的CaMKII簇。将培养的海马神经元暴露于无糖介质中的线粒体解偶联剂，可促进大量CaMKII免疫阳性结构的形成，这些结构在大小和形状上与亚细胞中观察到的CaMKII簇相同。CaMKII的聚集会阻止其接触固定底物，从而降低其激酶功能。另一方面，聚集不会影响CaMKII的大细胞池作为钙调蛋白宿的能力，正如金结合的钙调蛋白与CaMKII簇的钙结合所证明的那样。我们认为，观察到的CaMKII聚集成球形结构是一种保护性机制，可以防止在失去钙稳态时蛋白质过度磷酸化，而不会影响钙调蛋白的调节。
Cytoskeletal and postsynaptic density (PSD) fractions from forebrain contain discrete spherical structures that are immunopositive for Ca2+/calmodulin-dependent protein kinase II (CaMKII). Spherical structures viewed by rotary shadow electron microscopy have an average diameter of similar to 100 nm and, in distinction to postsynaptic densities, do not immunolabel for PSD-95. These structures were purified to near homogeneity by extraction with the detergent N-lauryl sarcosinate. Biochemical analysis revealed that CaMKII accounts for virtually all of the protein in the purified preparation, suggesting that spherical structures are clusters of self-associated CaMKII. Exposure of cultured hippocampal neurons to a mitochondrial uncoupler in glucose-free medium promotes the formation of numerous CaMKII-immunopositive structures identical in size and shape to the CaMKII clusters observed in subcellular fractions. Clustering of CaMKII would reduce its kinase function by preventing its access to fixed substrates. On the other hand, clustering would not affect the ability of the large cellular pool of CaMKII to act as a calmodulin sink, as demonstrated by the Ca2+-dependent binding of gold-conjugated calmodulin to CaMKII clusters. We propose that the observed clustering of CaMKII into spherical structures is a protective mechanism preventing excessive protein phosphorylation upon loss of Ca2+ homeostasis, without compromising calmodulin regulation.