Bernaudo Stefanie, Khazai Shahin, Honarparvar Eilyad, Kopteva Alina, Peng Chun
Department of Biology, York University, Toronto, Canada.
Centre for Research on Biomolecular Interactions, Faculty of Science, York University, Toronto, Canada.
PLoS One. 2017 Jun 22;12(6):e0179906. doi: 10.1371/journal.pone.0179906. eCollection 2017.
Cyclin G2 (CCNG2) is an atypical cyclin that functions to inhibit cell cycle progression and is often dysregulated in human cancers. We have previously shown that cyclin G2 is highly unstable and can be degraded through the ubiquitin/proteasome pathway. Furthermore, cyclin G2 contains a PEST domain, which has been suggested to act as a signal for degradation by multiple proteases. In this study, we determined if calpains, a family of calcium-dependent proteases, are also involved in cyclin G2 degradation. The addition of calpain inhibitors or silencing of calpain expression by siRNAs strongly enhanced cyclin G2 levels. On the other hand, incubation of cell lysates with purified calpains or increasing the intracellular calcium concentration resulted in a decrease in cyclin G2 levels. Interestingly, the effect of calpain was found to be dependent on the phosphorylation of cyclin G2. Using a kinase inhibitor library, we found that Epidermal Growth Factor (EGF) Receptor is involved in cyclin G2 degradation and treatment with its ligand, EGF, induced cyclin G2 degradation. In addition, the presence of the PEST domain is necessary for calpain and EGF action. When the PEST domain was completely removed, calpain or EGF treatment failed to trigger degradation of cyclin G2. Taken together, these novel findings demonstrate that EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2 and that the PEST domain is critical for EGF/calpain actions.
细胞周期蛋白G2（CCNG2）是一种非典型的细胞周期蛋白，其功能是抑制细胞周期进程，在人类癌症中常发生失调。我们之前已经表明，细胞周期蛋白G2高度不稳定，可通过泛素/蛋白酶体途径降解。此外，细胞周期蛋白G2含有一个PEST结构域，有人认为它可作为多种蛋白酶降解的信号。在本研究中，我们确定钙蛋白酶（一类钙依赖性蛋白酶）是否也参与细胞周期蛋白G2的降解。添加钙蛋白酶抑制剂或通过小干扰RNA沉默钙蛋白酶表达可强烈提高细胞周期蛋白G2水平。另一方面，用纯化的钙蛋白酶孵育细胞裂解物或提高细胞内钙浓度会导致细胞周期蛋白G2水平降低。有趣的是，发现钙蛋白酶的作用取决于细胞周期蛋白G2的磷酸化。使用激酶抑制剂文库，我们发现表皮生长因子（EGF）受体参与细胞周期蛋白G2的降解，用其配体EGF处理可诱导细胞周期蛋白G2降解。此外，PEST结构域的存在对于钙蛋白酶和EGF的作用是必需的。当PEST结构域被完全去除时，钙蛋白酶或EGF处理无法触发细胞周期蛋白G2的降解。综上所述，这些新发现表明，EGF诱导的、钙蛋白酶介导的蛋白水解作用有助于细胞周期蛋白G2的快速降解，并且PEST结构域对于EGF/钙蛋白酶的作用至关重要。
