Department of Molecular Genetics, Centre for Research on Agricultural Genomics CRAG, CSIC-IRTA-UAB, Barcelona, Spain.
PLoS One. 2011;6(7):e21909. doi: 10.1371/journal.pone.0021909. Epub 2011 Jul 15.
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 是一种高度多功能的丝氨酸/苏氨酸激酶，在真核生物中普遍存在。CK2 组织为异四聚体酶，由两种类型的亚基组成：催化（CK2α）和调节（CK2β）。CK2β 亚基增强了全酶的稳定性、活性和特异性，但它们也可以独立于 CK2 四聚体发挥功能。植物中的 CK2β 调节亚基与动物或酵母的对应物不同，因为它们具有大约 90 个氨基酸的额外特定的 N 端延伸，与任何以前表征的功能结构域都没有同源性。陆地植物 CK2β 亚基序列的 N 端结构域的序列分析揭示了其通过短的、保守的基序排列，其中一些包括 CK2 自身磷酸化位点。通过使用玉米 CK2β1 和缺失 N 端结构域的版本（ΔNCK2β1），我们已经证明 CK2β1 在 N 端结构域内自身磷酸化。此外，由 CK2α1/ΔNCK2β1 组成的全酶比 CK2α1/CK2β1 或 CK2α 单独更有效地磷酸化不同的底物。在不同的植物系统中瞬时过表达与 GFP 融合的 CK2β1 和 ΔNCK2β1 表明，存在 N 端结构域会增强核斑点中的聚集并稳定蛋白质免受蛋白酶体降解。最后，双分子荧光互补（BiFC）测定显示植物 CK2 全酶的核内和细胞质位置，与主要在核内观察到的单个 CK2α/β 亚基相反。总的来说，我们的结果支持 CK2β 亚基的植物特异性 N 端结构域参与 CK2 全酶活性的下调和 CK2β1 蛋白的稳定的假设。总之，这项工作中显示的全部数据表明，该结构域是植物为调节目的而获得的。
