Characterization of p53-independent ARF pathway

描述（由申请人提供）：ARF肿瘤抑制因子被广泛认为是p53响应超增殖信号的主要上游激活因子。然而，第二种不依赖p53的ARF通路的出现改变了我们对ARF肿瘤监测的看法。具体而言，动物模型提供了另一种ARF途径的遗传证据。缺乏ARF和p53的小鼠与单一缺乏ARF或p53的窝鼠相比，表现出更深刻的肿瘤谱，并且在缺乏ARF或ARF/p53的动物中观察到的眼睛缺陷在p53缺失的小鼠中未见。最近的实验进一步证明，在p53突变体存在或p53和Mdm2均不存在的情况下，ARF可诱导细胞生长停滞。我们的长期目标是了解ARF不依赖p53特性的潜在机制。为了寻找不依赖p53的ARF靶点，我们分离了一种新的ARF结合蛋白，核磷蛋白（NPM）。NPM是中心体的一个组成部分，也是细胞周期蛋白E-CDK2进入S期的关键磷酸化底物。以这种方式，磷酸化- npm作为中心体复制的关键调节因子。我们发现ARF-NPM复合物局限于核核，阻止NPM定位到中心体，而它通常作为细胞周期蛋白E-CDK2全酶的底物。此外，ARF阻止活性细胞周期蛋白Ecdk2复合物对NPM的直接磷酸化，抑制随后的中心体复制。重新引入Mdm2逆转了ARF不依赖p53的特性：NPM重新定位到中心体上，中心体复制，随后发生S期进展。我们假设ARF通过主动募集NPM进入核仁，远离周期蛋白E-cdk2复合物，可以作为p53独立的细胞周期抑制，抑制中心体复制的基本过程。

DESCRIPTION (provided by applicant): The ARF tumor suppressor is widely regarded as a major upstream activator of p53 in response to hyperproliferative signals. However, the emergence of a second, p53-independent ARF pathway has altered the way we think about ARF tumor surveillance. Specifically, animal models have provided genetic evidence of an alternative ARF pathway. Mice lacking ARF and p53 exhibit a more profound tumor spectrum when compared to single-null ARF or p53 littermates and eye defects observed in ARF or ARF/p53-null animals are not seen in p53-null mice. Recent experiments have further demonstrated that ARF can induce cell growth arrest in the presence of mutant p53, or in the absence of p53 and Mdm2 altogether. Our long-term objective is to understand the mechanisms underlying ARF's p53-independent properties. In search of p53-independent ARF targets, we isolated a novel ARF binding protein, nucleophosmin (NPM). NPM is a component of the centrosome and is also a critical phospho-substrate for cyclin E-CDK2 during progression into S phase. In this manner, phospho-NPM serves as a key regulator of centrosome duplication. We have found that the ARF-NPM complex is restricted to the nucleolus, preventing NPM localization to the centrosome where it normally serves as a substrate for cyclin E-CDK2 holoenzymes. Furthermore, ARF prevents the direct phosphorylation of NPM by active cyclin Ecdk2 complexes, inhibiting subsequent centrosome duplication. Re-introduction of Mdm2 reverses the p53-independent properties of ARF: NPM re-localizes to the centrosome, centrosomes duplicate, and S phase progression ensues. We hypothesize that ARF, through active recruitment of NPM into the nucleolus away from cyclin E-cdk2 complexes, can function as a p53-independent cell cycle brake, inhibiting the basic process of centrosome duplication.