Cloning, characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin‐dependent protein kinase Iγ in the rat brain
Cloning, characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin‐dependent protein kinase Iγ in the rat brain
复制标题
DOI：
10.1046/j.1471-4159.2003.01760.x
复制
发表时间：
2003-06
影响因子：
4.7
通讯作者：
H. Nishimura;H. Sakagami;Akiyoshi Uezu;K. Fukunaga;Makoto Watanabe;H. Kondo
中科院分区：
文献类型：
--
作者：
H. Nishimura;H. Sakagami;Akiyoshi Uezu;K. Fukunaga;Makoto Watanabe;H. Kondo
Ca2+/钙调蛋白依赖性蛋白激酶I (CaMKI)，最初被鉴定为磷酸化突触蛋白I的蛋白激酶，已被证明构成一个密切相关的亚型家族（α， β和γ）。在这里，我们分离并确定了CaMKI的两个选择性剪接异构体CaMKIγ1和‐γ2的完整初级结构。由于camki - γ - 1在camki - γ - 2中缺失了425 bp的核苷酸序列，camki - γ - 1和‐γ - 2具有相同的N端催化结构域和不同的C端催化区域。体外激酶实验表明，与Ca2+/钙调素依赖性蛋白激酶（CaMKK）预孵育后，CaMKIγ1的Ca2+/CaM依赖性活性显著增强，但CaMKIγ2没有明显活化。Northern blot分析显示CaMKIγ在大脑中主要表达。RT‐PCR分析显示camki - γ1和camki - γ2在大脑不同区域的表达模式相似。原位杂交分析表明，CaMKIγ mRNA的表达模式与其他CaMKI亚型不同，主要表达于一些受限制的大脑区域，如嗅球、海马锥体CA3细胞层、中央杏仁核、下丘脑腹内侧核和松果体。在海马原代神经元和NG108 - 15细胞中，转染的camki - γ - 1和‐γ - 2主要定位于细胞质和神经突，而不在细胞核中。这些发现表明，CaMKIγ的两种亚型都可能参与某些神经元细胞胞质室的Ca2+信号转导。
Ca2+/calmodulin‐dependent protein kinase I (CaMKI), originally identified as a protein kinase phoshorylating synapsin I, has been shown to constitute a family of closely related isoforms (α, β and γ). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKIγ1 and ‐γ2. CaMKIγ1 and ‐γ2 contain an identical N‐terminal catalytic domain with different C‐terminal regions due to the deletion of the 425‐bp nucleotide sequence of CaMKIγ1 in CaMKIγ2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+/CaM‐dependent activity of CaMKIγ1 by the preincubation with Ca2+/calmodulin‐dependent protein kinase kinase (CaMKK), but no significant activation of CaMKIγ2. Northern blot analysis has demonstrated the predominant expression of CaMKIγ in the brain. RT‐PCR analysis has revealed similar expression patterns between CaMKIγ1 and CaMKIγ2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKIγ mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108‐15 cells, transfected CaMKIγ1 and ‐γ2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKIγ may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.