Moreno-Bueno G, Calés C, Behrens M M, Fernández-Renart M
Instituto de Investigaciones Biomédicas 'Alberto Sols', UAM-CSIC, C/ Arturo Duperier 4, 28029 Madrid, Spain.
Biochem J. 2000 Jul 15;349(Pt 2):527-37. doi: 10.1042/0264-6021:3490527.
In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase (CK)I from Dictyostelium discoideum is reported. The predicted amino acid sequence shares 70% identity with the catalytic domain of the mammalian delta and epsilon isoforms, Drosophila CKIepsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis. The level of DdCKI expression did not change during the cell cycle. Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D. discoideum extracts. Protein expression paralleled the pattern found for the RNA. The expression of DdCKI in Escherichia coli resulted in an active enzyme that autophosphorylated and phosphorylated casein. Immunofluorescence assays showed that DdCKI was localized in the cytoplasm and nuclei of Dictyostelium cells. The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D. discoideum. Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair.
在本研究中，报道了来自盘基网柄菌的一种49 kDa形式的酪蛋白激酶（CK）I的分子克隆及特性。预测的氨基酸序列与哺乳动物δ和ε亚型、果蝇CKIε和粟酒裂殖酵母Hhp1的催化结构域具有70%的同一性，与参与DNA修复的57 kDa形式的酵母CK即Hrr25具有63%的同一性。通过Northern印迹分析检测到，盘基网柄菌CKI（DdCKI）在营养期非同步细胞以及分化细胞中均有表达。DdCKI的表达水平在细胞周期中没有变化。针对该蛋白截短版本产生的抗体识别出了来自盘基网柄菌提取物的一种49 kDa蛋白。蛋白质表达与RNA的表达模式一致。DdCKI在大肠杆菌中的表达产生了一种具有自磷酸化和磷酸化酪蛋白活性的酶。免疫荧光分析表明，DdCKI定位于盘基网柄菌细胞的细胞质和细胞核中。CKI基因缺失突变体的缺乏表明该蛋白对盘基网柄菌的营养生长至关重要。DdCKI的过表达导致细胞对羟基脲的抗性增加，表明该激酶在DNA修复中可能发挥作用。
