Pinontoan Reinhard, Yuasa Takashi, Anderca Marinela I, Matsuoka Takashi, Uozumi Nobuyuki, Mori Hitoshi, Muto Shoshi
Nagoya University Bioscience Center, Chikusa-ku, Nagoya, 464-8601 JapanGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan Nagoya University Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 JapanGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan Nagoya University Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan.
J Phycol. 2000 Jun;36(3):545-552. doi: 10.1046/j.1529-8817.2000.99185.x. Epub 2001 Dec 25.
A cDNA clone encoding a Ca +-dependent protein kinase (DtCPK1) with a calculated molecular mass of 65,746 Da was isolated by sequential immuno- and hybridization-screening from a cDNA library of the halotolerant green alga, Dunaliella tertiolecta Butcher (Chlorophyceae). Primary structure analysis of DtCPK1 revealed a long variable domain preceding a catalytic domain, an autoinhibitory junction domain, and a C-terminal calmodulin-like domain containing 4 EF-hand motifs. Database searches showed that DtCPK1 has a high similarity to CCK1, a CDPK from the green alga, Chlamydomonas eugamentos Moewus. The N-terminal long variable domain of DtCPK1 contains neither the N-myristoylation motif, which is found in many CDPKs, nor the PEST motif, which is associated with rapid protein turnover and found in one CDPK subfamily. However, a putative Ca +-dependent lipid binding domain that might be responsible for the association of cytosolic DtCPK1 with the cell membrane was identified in the variable domain. Three CDPKs, with molecular masses of 62, 54, and 47 kDa respectively, were observed in an in-gel protein kinase assay of D. tertiolecta cells extract. No change in the activities of these CDPKs were observed for up to 30 min after D. tertiolecta cells had been subjected to a hypoosmotic shock. An antibody raised against a CDPK purified from D. tertiolecta and used to isolate the DtCPK1 cDNA clone cross-reacted strongly with the 62-kDa CDPK but weakly with the 54-kDa CDPK in a Western blot, indicating that the 62-kDa CDPK is identical to DtCPK1. There was no change in the intensity of these bands after hypoosmotic shock, implying that the cellular level of the enzyme protein is not associated with hypoosmotic shock. These results indicate that CDPK is activated only by the increase in cytosolic-free Ca + concentration in vivo.
通过免疫筛选和杂交筛选，从耐盐绿藻杜氏盐藻（绿藻纲）的cDNA文库中分离出一个编码钙依赖性蛋白激酶（DtCPK1）的cDNA克隆，其计算分子量为65,746道尔顿。DtCPK1的一级结构分析显示，在催化结构域之前有一个长的可变结构域、一个自抑制连接结构域以及一个含有4个EF手基序的C端钙调蛋白样结构域。数据库搜索表明，DtCPK1与来自绿藻莱茵衣藻的CDPK CCK1高度相似。DtCPK1的N端长可变结构域既不包含许多CDPK中发现的N-肉豆蔻酰化基序，也不包含与快速蛋白质周转相关且在一个CDPK亚家族中发现的PEST基序。然而，在可变结构域中鉴定出一个假定的钙依赖性脂质结合结构域，它可能负责胞质DtCPK1与细胞膜的结合。在杜氏盐藻细胞提取物的凝胶内蛋白激酶测定中，观察到三种分子量分别为62、54和47 kDa的CDPK。在杜氏盐藻细胞受到低渗冲击后长达30分钟内，这些CDPK的活性没有变化。一种针对从杜氏盐藻中纯化的CDPK制备的抗体，用于分离DtCPK1 cDNA克隆，在蛋白质免疫印迹中与62 kDa的CDPK强烈交叉反应，而与54 kDa的CDPK弱交叉反应，表明62 kDa的CDPK与DtCPK1相同。低渗冲击后这些条带的强度没有变化，这意味着酶蛋白的细胞水平与低渗冲击无关。这些结果表明，CDPK在体内仅通过胞质游离钙浓度的增加而被激活。
