Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
Biochem Biophys Res Commun. 2021 Apr 9;548:98-103. doi: 10.1016/j.bbrc.2021.02.064. Epub 2021 Feb 25.
E1A is an adenoviral protein which is expressed at the early phase after viral infection and contains four conserved regions (CR1, CR2, CR3 and CR4). Our previous work suggests that E1A facilitates the formation of cyclin A-CDK2 complex and thereby enhances CDK2 activity. However, the molecular function of E1A in CDK2 activation has been unclear. Here, we studied the mechanism of enhancement of CDK2 activity by E1A, using the E1A variant forms which selectively contain CR domains. We isolated four E1A variant forms, i.e. 13S (containing CR1, CR2, CR3, CR4), 12S (CR1, CR2, CR4), 10S (CR2, CR4) and 9S (CR4), derived from HEK293 cells which express E1A. 13S promoted G2/M-phase arrest, upon CDK2 hyper-activation by co-expressing a stabilized cyclin A mutant, most strongly among those E1A variant forms. Concomitantly, the specific activity of the 13S-associated CDK2 was highest among them. 10S exhibited lower affinity for CDK2 than the 13S while the affinity for CDK2 was comparable between 13S and 12S. Nonetheless, 12S did not enhance the CDK2 specific activity. On the other hand, a mutation in CR2 domain, which is essential for binding to p107, suppressed both the binding and activation of CDK2. These results suggest that CR1 domain, in addition to CR2 domain via p107 interaction, is important for binding to CycA-CDK2 complex while CR3 domain facilitates CDK2 activation. Since the function of CR3 in cell cycle regulation has been relatively unknown, we propose the enhancement of CDK2 activity as a novel function of CR3 domain.
E1A 是一种腺病毒蛋白，在病毒感染后早期表达，包含四个保守区域（CR1、CR2、CR3 和 CR4）。我们之前的工作表明，E1A 有助于形成细胞周期蛋白 A-CDK2 复合物，从而增强 CDK2 活性。然而，E1A 激活 CDK2 的分子功能尚不清楚。在这里，我们使用选择性包含 CR 结构域的 E1A 变体形式研究了 E1A 增强 CDK2 活性的机制。我们从表达 E1A 的 HEK293 细胞中分离出四种 E1A 变体形式，即 13S（包含 CR1、CR2、CR3、CR4）、12S（CR1、CR2、CR4）、10S（CR2、CR4）和 9S（CR4）。在共表达稳定化的 cyclin A 突变体使 CDK2 过度激活的情况下，13S 促进 G2/M 期阻滞最为强烈，在这些 E1A 变体形式中。同时，它们的 CDK2 相关的特异性活性最高。10S 与 13S 相比，对 CDK2 的亲和力较低，而 13S 和 12S 之间的 CDK2 亲和力相当。尽管如此，12S 并没有增强 CDK2 的特异性活性。另一方面，CR2 结构域中的突变，对于与 p107 的结合是必需的，抑制了 CDK2 的结合和激活。这些结果表明，除了通过 p107 相互作用的 CR2 结构域外，CR1 结构域对于与 CycA-CDK2 复合物的结合很重要，而 CR3 结构域促进 CDK2 的激活。由于 CR3 结构域在细胞周期调控中的功能相对未知，我们提出增强 CDK2 活性是 CR3 结构域的一个新功能。
