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

从杜氏藻的可溶性部分中纯化了2万倍的钙依赖蛋白激酶(CDPK)，并对其进行了鉴定。利用抗杜氏杆菌CDPK抗体，从杜氏藻基因文库中克隆了编码部分CDPK基因的cDAN。以部分cDNA为探针，扩增出全长cDNAs，并测定了其核苷酸序列。从核苷酸序列推定的氨基酸序列表明，杜氏藻CDPK是一个典型的CDPK，其N端有激活域，C端有钙调蛋白样域，它们之间有假底物结构域。当受到低/高渗休克时，具有不同底物特异性和激活时间的40 kDa蛋白激酶分别被激活，称为HAP/LAP激酶。这种激活伴随着蛋白质的磷酸化。由于细胞内Ca~(2+)和Gt~(2+)的增加预计会参与激活，转化Aequorin的烟草悬浮培养细胞受到渗透休克。低渗休克时，细胞内Ca~(2+)和Gt~(2+)有一过性升高，峰值出现在休克后70s，随后50、75和80 kDa蛋白激酶被激活。在微咸水轮藻中检测到CDPK与抗Dunaliella CDPK的交叉反应。当该藻将抗体注入细胞质受到低渗休克时，即使休克后胞内Ca~(2+)~(2+)增加，膨压调节也受到抑制。本研究结果表明，CDPK与藻类和高等植物的渗透压信号转导和渗透调节密切相关。

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.