Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria
Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria
复制标题
DOI：
10.1186/1746-4811-8-23
复制
发表时间：
2012-06-28
期刊：
影响因子：
5.1
通讯作者：
Schnittger, Arp
中科院分区：
文献类型：
--
作者：
Harashima, Hirofumi;Schnittger, Arp
背景：细胞增殖是植物生长发育的重要决定因素。此外，细胞分裂速率的调节是植物可塑性的重要机制，是植物适应环境条件的关键。了解开花植物细胞周期的最大挑战之一是CDK和细胞周期蛋白的大家族，它们有可能形成许多不同的复合物。然而，很大程度上不清楚哪些复合物是活性的。此外，还有许多CDK和细胞周期蛋白相关蛋白，其生物学作用仍不清楚，即它们是否确实具有酶活性。因此，这些蛋白质的生化特性是理解其function.Results的关键重要性：在这里，我们提出了一个简单的系统，系统地表达和纯化活性CDK-cyclin复合物从大肠杆菌。大肠杆菌提取物。我们的方法依赖于伴随产生的CDK活化激酶，其催化激酶活性所必需的T环磷酸化。以G1期细胞周期蛋白CYCLIN D3;1（CYCD 3;1）、有丝分裂细胞周期蛋白CYCLIN B1;2（CYCB 1;2）和非典型减数分裂细胞周期蛋白SOLO DANCERS（SDS）与A型、B1型和B2型CDKs结合为例，我们发现不同的CDKs可以在体外与各种细胞周期蛋白相互作用，但只有少数特异性复合物具有高水平的激酶活性。我们的工作表明，细胞周期蛋白以及CDK伙伴有助于底物特异性在植物中。这些发现完善了细胞周期控制中的相互作用网络，并确定了在育种中调节细胞增殖活性的特定复合物。
Background: Cell proliferation is an important determinant of plant growth and development. In addition, modulation of cell-division rate is an important mechanism of plant plasticity and is key in adapting of plants to environmental conditions. One of the greatest challenges in understanding the cell cycle of flowering plants is the large families of CDKs and cyclins that have the potential to form many different complexes. However, it is largely unclear which complexes are active. In addition, there are many CDK- and cyclin-related proteins whose biological role is still unclear, i.e. whether they have indeed enzymatic activity. Thus, a biochemical characterization of these proteins is of key importance for the understanding of their function.Results: Here we present a straightforward system to systematically express and purify active CDK-cyclin complexes from E. coli extracts. Our method relies on the concomitant production of a CDK activating kinase, which catalyzes the T-loop phosphorylation necessary for kinase activity. Taking the examples of the G1-phase cyclin CYCLIN D3;1 (CYCD3;1), the mitotic cyclin CYCLIN B1;2 (CYCB1;2) and the atypical meiotic cyclin SOLO DANCERS (SDS) in conjunction with A-, B1- and B2-type CDKs, we show that different CDKs can interact with various cyclins in vitro but only a few specific complexes have high levels of kinase activity.Conclusions: Our work shows that both the cyclin as well as the CDK partner contribute to substrate specificity in plants. These findings refine the interaction networks in cell-cycle control and pinpoint to particular complexes for modulating cell proliferation activity in breeding.