Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish
Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish
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DOI：
10.1016/j.abb.2006.09.034
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发表时间：
2007-01-15
影响因子：
3.9
通讯作者：
Kameshita, Isamu
中科院分区：
文献类型：
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作者：
Nimura, Takaki;Sueyoshi, Noriyuki;Kameshita, Isamu
核 Ca2+/钙调蛋白依赖性蛋白激酶磷酸酶 (CaMKP-N) 是一种在体外去磷酸化并同时下调三功能 Ca2+/钙调蛋白依赖性蛋白激酶 (CaMK) 的酶。然而，这种酶在体内的功能作用尚不清楚。为了研究 CaMKP-N 在斑马鱼胚胎发生过程中的生物学意义，我们克隆并表征了斑马鱼 CaMKP-N (zCaMKP-N)。根据斑马全基因组Shotgun数据库中的核苷酸序列，我们分离出zCaMKP-N的cDNA克隆，其编码633个氨基酸残基的蛋白质。小鼠神经母细胞瘤 Neuro2a 细胞中瞬时表达的全长 zCaMKP-N 被发现位于细胞核中。相反，缺乏RKKRRLDVLPLRR（残基575-587）的C端截短突变体具有细胞质染色，表明zCaMKP-N的核定位信号存在于C端区域。 CaMKIV 转染 Neuro2a 细胞的离子霉素处理导致 CaMKIV 磷酸化形式显着增加。然而，与 zCaMKP-N 共转染显着降低了离子霉素刺激细胞中的磷酸化 CaMKP-N。斑马鱼胚胎的整体镶嵌杂交分析表明，zCaMKP-N 仅在头部和神经管区域表达。使用基于吗啉基的反义寡核苷酸对 zCaMKP-N 进行基因敲除可诱导斑马鱼胚胎显着的形态异常。在异常胚胎的脑和脊髓中观察到大量凋亡细胞。这些结果表明 zCaMKP-N 在斑马鱼的早期发育中起着至关重要的作用。 (c) 2006 Elsevier Inc. 保留所有权利。
Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates triultifunctional Ca2+/Calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebratish whole genome Shotgun database, we isolated a cDNA clone For zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in Mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. lonomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKP-N in ionomycin-stimulated cells. Whole mount in siru hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebratish. (c) 2006 Elsevier Inc. All rights reserved.