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Calcium-dependent protein kinase in osmoregulation of the halotolerant green alga Dunaliella tertiolecta

钙依赖性蛋白激酶在耐盐绿藻 Dunaliella tertiolecta 渗透调节中的作用

基本信息

批准号：
06454013
负责人：
TORIYAMA Shoshi
金额：
$ 4.29万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Calcium-dependent protein kinase (CDPK)was purified 20,000-fold from soluble franction of Dunaliella, and characterized. Using an anti-Dunaliella CDPK antibody, cDANs encoding parts of CDPK were cloned from a Dunaliella cDNA library. A full length cDNA was isolated by using one of partial cDNAs as a probe, and is nucleotide sequence was determined. Deduced amino acid squence from the nucleotide sequence indicated that Dunaliella CDPK in a typical CDPK which has kinase domain at N-terminus, calmolulin-like domain at C-terminus and pseudosubstrate domain between them.When subjected to hypo-/hypersomotic shock, 40-kDa protein kinases named HAP/LAP kinases, respectively, which have different substrate specificities and time courses of activation were activated. The activation accompanied with protein phosphorylation. Since an involvement of increase in cytosolic Ca^<2+> was anticipated for the activation, tobacco suspension culture cells transformed aequorin was subjected osmotic shock. Upon hypoosmotic shock, a transient increase in Ca^<2+> with a peak at 70 sec after the shock and subsequent activation of 50-, 75- and 80-kDa protein kinases was observed. The activation accompanied with protein phosphorylation suggesting the involvement of CDPK.A CDPK cross-reacting with anti-Dunaliella CDPK was detected in a brackish water Characeae Lamprothamnium. When this alga injected the antibody into the cytoplasm was subjected to hypoosmotic shock, turgor regulation was suppressed even though an increase in cytosolic Ca^<2+> occured after the shock. This result suggests the involvement of CDPK in turgor regulation of this alga.The results obtained in this study suggest that CDPK is tightly involved in osmotic signal transduction and osmoregulation in algae and higher plants.

从杜氏藻的可溶性组分中纯化了 20,000 倍的钙依赖性蛋白激酶 (CDPK)，并进行了表征。使用抗杜氏藻 CDPK 抗体，从杜氏藻 cDNA 文库中克隆编码 CDPK 部分的 cDAN。使用部分cDNA之一作为探针分离全长cDNA，并确定其核苷酸序列。从核苷酸序列推导的氨基酸序列表明，杜氏藻CDPK是典型的CDPK，N端有激酶结构域，C端有类钙调蛋白结构域，两者之间有假底物结构域。当受到低/高睡眠休克时，40 kDa的蛋白激酶分别命名为HAP/LAP激酶，它们具有不同的底物激活的特异性和时间过程被激活。激活伴随着蛋白质磷酸化。由于预计活化涉及胞质Ca 2+ 的增加，因此对转化的水母发光蛋白的烟草悬浮培养细胞进行渗透休克。低渗休克后，观察到Ca 2+ 短暂增加，在休克后70秒达到峰值，并随后激活50-、75-和80-kDa蛋白激酶。激活伴随着蛋白质磷酸化，这表明 CDPK 的参与。在咸水中检测到了 CDPK 与抗杜氏藻 CDPK 的交叉反应。当将抗体注射到细胞质中的该藻类受到低渗休克时，尽管休克后细胞溶质Ca 2+ 增加，但膨压调节受到抑制。这一结果表明CDPK参与该藻类的膨压调节。本研究获得的结果表明CDPK紧密参与藻类和高等植物的渗透信号转导和渗透调节。