Inhibition of APC-mediated proteolysis by the meiosis-specific protein kinase Ime2

Proteolysis triggered by the anaphase-promoting complex (APC) is needed for sister chromatid separation and the exit from mitosis. APC is a ubiquitin ligase whose activity is tightly controlled during the cell cycle. To identify factors involved in the regulation of APC-mediated proteolysis, a Saccharomyces cerevisiae GAL-cDNA library was screened for genes whose overexpression prevented degradation of an APC target protein, the mitotic cyclin Clb2. Genes encoding G(1), S, and mitotic cyclins were identified, consistent with previous data showing that the cyclin-dependent kinase Cdk1 associated with different cyclins is a key factor for inhibiting APC(Cdh1) activity from late-G(1) phase until mitosis. In addition, the meiosis-specific protein kinase Ime2 was identified as a negative regulator of APC-mediated proteolysis. Ectopic expression of IME2 in G(1) arrested cells inhibited the degradation of mitotic cyclins and of other APC substrates. IME2 expression resulted in the phosphorylation of Cdh1 in G(1) cells, indicating that Ime2 and Cdk1 regulate APC(Cdh1) in a similar manner. The expression of IME2 in cycling cells inhibited bud formation and caused cells to arrest in mitosis. We show further that Ime2 itself is an unstable protein whose proteolysis occurs independently of the APC and SCF (Skp1/Cdc53/F-box) ubiquitin ligases. Our findings suggest that Ime2 represents an unstable, meiosis-specific regulator of APC(Cdh1).

由后期促进复合物（anaphase-promoting complex, APC）介导的蛋白水解作用，是姐妹染色单体分离与有丝分裂退出所必需的过程。APC是一种泛素连接酶，其活性在细胞周期中受到严格调控。为鉴定参与APC介导的蛋白水解调控的因子，研究人员以酿酒酵母（Saccharomyces cerevisiae）的GAL-cDNA文库为材料，筛选过表达可阻断APC靶蛋白——有丝分裂周期蛋白Clb2——降解的基因。研究鉴定出编码G1期、S期及有丝分裂周期蛋白的基因，这与既往研究结论一致：与不同周期蛋白结合的细胞周期蛋白依赖性激酶Cdk1（cyclin-dependent kinase Cdk1），是晚G1期至有丝分裂阶段抑制APC(Cdh1)活性的关键调控因子。此外，研究还鉴定出减数分裂特异性蛋白激酶Ime2为APC介导的蛋白水解的负调控因子。在G1期阻滞细胞中异位表达IME2，可抑制有丝分裂周期蛋白及其他APC底物的降解。在G1期细胞中，IME2的表达会诱导Cdh1发生磷酸化，这表明Ime2与Cdk1以相似的方式调控APC(Cdh1)的活性。在增殖细胞中表达IME2，会抑制出芽形成并使细胞阻滞于有丝分裂阶段。我们进一步证实，Ime2本身是一种不稳定蛋白，其蛋白水解过程不依赖于APC与SCF（Skp1/Cdc53/F-box）泛素连接酶。本研究结果表明，Ime2是一类不稳定的、减数分裂特异性的APC(Cdh1)调控因子。