Degradation of the CDK inhibitor p27^<Kip1> by a novel ubiquitin ligase.

细胞周期进程是由周期蛋白依赖性激酶（CDKs）控制的。CDK活性受CDK抑制蛋白（CKIs）的负调控。在静止期和G1期早期，Cip/ kip型CKIs之一p27^<Kip1>在细胞核中大量存在，抑制细胞周期进程。p27^<Kip1>在G1期晚期通过泛素-蛋白酶体途径被降解，使细胞进入S期。在这项研究中，我们发现p27NBP1是一个p27^<Kip1>-相互作用蛋白。p27NBP1与p27^<Kip1>相互作用，并以完整的环指结构域依赖的方式直接泛素化p27^<Kip1>。由于我们将p27NBP1分离为一种与含有Ser10的p27^<Kip1>的氨基末端相互作用的蛋白质，我们接下来研究了这两种蛋白质之间的相互作用是否受到Ser10磷酸化状态的影响。尽管替换了Ser10， p27^<Kip1>突变体与p27NBP1的关联程度与野生型p27^<Kip1>相似。这一结果表明p27NBP1与p27^<Kip1>相互作用，与Ser10磷酸化状态无关。小干扰RNA消融内源性p27NBP1增加了p27^<Kip1>的稳态水平，减缓了p27^<Kip1>的转换。p27^<Kip1>在G1晚期随着p27NBP1的积累同步减少。此外，p27NBP1的缺失以不依赖p53的方式抑制细胞周期从G1期到S期的进展。总的来说，结果表明p27NBP1通过促进泛素依赖性蛋白酶体降解，作为p27^<Kip1>功能的负调节因子。

Cell cycle progression is controlled by cyclin-dependent kinases (CDKs). The CDK activities are negatively regulated by CDK inhibitor proteins (CKIs). In the quiescent and early G1 phase, p27^<Kip1>, one of the Cip/Kip-type CKIs, exists in abundance in the cell nucleus to suppress cell cycle progression. p27^<Kip1> is degraded in late G1 phase by the ubiquitin-proteasome pathway, allowing cells to enter S phase. In this study, we identified p27NBP1 as a p27^<Kip1>-interacting protein. p27NBP1 physically interacted with p27^<Kip1> and directly ubiquitinated p27^<Kip1> in an intact RING finger domain-dependent manner. Since we isolated p27NBP1 as a protein that interacts with the amino-terminal portion of p27^<Kip1> containing Ser10, we next examined whether interaction between these two proteins was affected by Ser10 phosphorylation status. In spite of the substitution of Ser10, p27^<Kip1> mutants associated with p27NBP1 to an extent similar to that of wild-type p27^<Kip1>. This result suggested that p27NBP1 interacts with p27^<Kip1> regardless of the status of Ser10 phosphorylation. Ablation of endogenous p27NBP1 by small interfering RNA increased the steady-state level of p27^<Kip1> and decelerated p27^<Kip1> turnover. p27^<Kip1> was decreased in synchronization with accumulation of p27NBP1 in late G1 phase. Furthermore, depletion of p27NBP1 resulted in inhibition of cell cycle progression from G1 to S phase in a p53-independent manner. Overall, the results indicate that p27NBP1 acts as a negative regulator of p27^<Kip1> function by promoting ubiquitin-dependent proteasomal degradation.