Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes.

蛋白激酶CK2（Protein kinase CK2）是一类在真核生物中广泛分布的多功能丝氨酸/苏氨酸激酶（Ser/Thr kinase）。CK2以异四聚体全酶形式存在，由两种亚基构成：催化亚基（CK2α）与调节亚基（CK2β）。CK2β亚基可提升全酶的稳定性、活性与底物特异性，同时也能脱离CK2四聚体独立行使功能。植物来源的CK2β调节亚基与动物、酵母中的同源物存在显著差异，其带有一段约90个氨基酸的植物特异性N端延伸区域，该区域与所有已鉴定的功能域均无序列同源性。对陆生植物CK2β亚基的N端结构域进行序列分析后发现，该区域由短而保守的基序串联排列而成，其中部分基序包含CK2自身磷酸化位点。本研究以玉米CK2β1及其缺失N端结构域的截短变体（ΔNCK2β1）为实验材料，证实CK2β1的自身磷酸化发生于N端结构域内部。此外，由CK2α1与ΔNCK2β1组装形成的全酶，相比CK2α1/CK2β1全酶或单独的CK2α，能够更高效地磷酸化多种底物。将CK2β1与ΔNCK2β1分别与绿色荧光蛋白（Green Fluorescent Protein, GFP）融合后，在不同植物系统中开展瞬时过表达实验，结果显示N端结构域的存在可促进亚基在核斑中聚集，并增强该蛋白抵抗蛋白酶体降解的能力。最后，双分子荧光互补（Bimolecular Fluorescence Complementation, BiFC）实验表明，植物CK2全酶定位于细胞核与细胞质中；而单独的CK2α或CK2β亚基则主要定位于细胞核，二者的亚细胞定位模式存在显著差异。综合所有实验结果，本研究支持如下假说：植物特异性的CK2β亚基N端结构域参与下调CK2全酶的催化活性，并对CK2β1蛋白起到稳定作用。综上，本研究的全部实验数据表明，该N端结构域是植物为实现调控功能而获得的特有结构。