Characterization of p53-independent ARF pathway

描述(申请人提供)：ARF肿瘤抑制因子被广泛认为是P53的主要上游激活物，以响应过度增殖的信号。然而，第二个非p53依赖的ARF途径的出现改变了我们对ARF肿瘤监测的看法。具体地说，动物模型已经提供了替代ARF途径的遗传学证据。与单一缺失的ARF或p53相比，缺乏ARF和P53的小鼠表现出更深刻的肿瘤谱，并且在ARF或ARF/P53缺失的动物中观察到的眼睛缺陷在P53缺失的小鼠中看不到。最近的实验进一步证明，ARF可以在突变型P53存在的情况下或在P53和MDM2完全缺失的情况下诱导细胞生长停滞。我们的长期目标是了解ARF的P53非依赖性特性背后的机制。为了寻找非p53依赖的ARF靶点，我们分离了一种新的ARF结合蛋白--核磷蛋白(NPM)。NPM是中心体的组成部分，也是细胞周期蛋白E-CDK2进入S期的关键磷酸底物。通过这种方式，磷酸化NPM作为中心体复制的关键调节因子。我们发现ARF-NPM复合体仅限于核仁，阻止了NPM定位到中心体，而中心体通常是细胞周期蛋白E-CDK2全酶的底物。此外，ARF通过激活细胞周期蛋白Ecdk2复合体阻止NPM的直接磷酸化，抑制随后的中心体复制。MDM2的重新引入逆转了ARF的P53非依赖性特性：NPM重新定位到中心体，中心体重复，随后发生S期进展。我们推测，ARF通过主动地将NPM募集到远离细胞周期蛋白E-CDK2复合体的核仁中，发挥不依赖于P53的细胞周期刹车的作用，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.