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对过度增殖信号应答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，动物模型提供了替代ARF途径的遗传证据。与单一空ARF或p53同窝小鼠相比，缺乏ARF和p53的小鼠表现出更深的肿瘤谱，并且在ARF或ARF/p53空小鼠中观察到的眼缺陷在p53空小鼠中未观察到。最近的实验进一步证明，ARF可以在突变型p53存在下或在p53和Mdm 2完全不存在下诱导细胞生长停滞。我们的长期目标是了解ARF的p53非依赖性的机制。在寻找p53非依赖性ARF靶点的过程中，我们分离了一种新的ARF结合蛋白--核磷蛋白（NPM）。NPM是中心体的组成部分，也是细胞周期蛋白E-CDK 2进入S期的关键磷酸化底物。以这种方式，磷酸-NPM作为中心体复制的关键调节因子。我们已经发现，ARF-NPM复合物仅限于核仁，阻止NPM定位到中心体，在那里它通常作为细胞周期蛋白E-CDK 2全酶的底物。此外，ARF阻止NPM的直接磷酸化的活性细胞周期蛋白Ecdk 2复合物，抑制随后的中心体复制。重新引入Mdm 2逆转了ARF的p53非依赖性特性：NPM重新定位于中心体，中心体复制和S期进展终止。我们假设，ARF，通过积极招聘NPM进入核仁远离细胞周期蛋白E-cdk 2复合物，可以作为一个p53-独立的细胞周期制动器，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.