CrpF46(centrosome-related protein, F46)是本课题组发现的一种新的中心体蛋白，前期工作已表明CrpF46的亚细胞定位具有细胞周期依赖性：在间期，可观察到其特异定位在中心体上，而在分裂期，该蛋白又弥散分布在细胞质当中；另外还发现当它低表达时会出现多极纺锤体并扰乱细胞迁移过程中细胞的极性。为进一步研究CrpF46的功能，我们决定利用酵母双杂交技术从HeLa细胞cDNA文库中筛选出能够与其直接相互作用的蛋白质。将转化了CrpF46基因的酵母细胞AH109和含有HeLa细胞cDNA文库的Y187菌株杂交后，我们把杂交产物涂在酵母缺陷培养基上进行培养，经过3轮筛选和回转杂交后最终筛选出11种能够在酵母细胞中与CrpF46发生相互作用的候选蛋白，其中包括PCNA、SBF2、HECTD1、SNAPAP和TRIM2等。分析了这些蛋白质在细胞中的主要作用后，我们决定选择其中在有丝分裂过程中处于细胞周期检验点中心位置的增殖细胞核抗原（PCNA）进行进一步的研究。我们利用免疫共沉淀（Co-IP）实验确定了在HeLa细胞中CrpF46与PCNA依然能够相互作用，间接免疫荧光实验结果表明这两种蛋白在细胞质中有比较明显的共定位，因此推断这两个蛋白可能是在细胞质中发生互作。另外，我们将CrpF46的3个卷曲螺旋（coiled-coil）结构域分别重组到原核表达载体中以进行融合蛋白沉降（Pull-down）实验，初步确定了CrpF46与PCNA互作的序列位于1~241 aa之间（第一个coiled-coil结构域）。同时，我们还发现CrpF46与PCNA的结合蛋白cyclin A和cyclin E以及抑癌蛋白p21和p53能够形成复合体，因此我们推测CrpF46参与了PCNA调节细胞周期的作用网络。为了进一步确定CrpF46在细胞周期相关生理功能，我们计划接下来研究p21和p53对CrpF46的功能是否具有调节作用，并确定PCNA与CrpF46相互作用的具体时期等。

Centrosome related protein F46 (CrpF46) is a novel protein discovered by our group a few years ago. We found the cell cycle dependent subcellular localization of this protein. In the interphase of a cell cycle, it specially localizes on the centrosomes, though during the mitotic phase, it spreads around the cytoplasm. After CrpF46 silenced by transfecting antisence RNA into HeLa cells, the growth, polarity and movement of the cells were affected, appearing multipolar spindles and then resulting in cell apotosis.To further research the functions of CrpF46, we decided to seek its direct interacting proteins by the means of Yeast Two Hybrid Systerm. After transfecting AH109 yeast cells by recombinant CrpF46 gene, we make these cells hybrid with Y187 yeast cells transfected with HeLa cDNA library, then cultured the hybidization products on specific mediums. Through screening, we finally obtained 11 proteins from over two thousand of clones, including PCNA, SBF2, HECTD1, SNAPAP, TRIM2 and others. Amongst these 11 proteins, we were very interested in PCNA, proliferating cell nuclear antigen, so we identified the interaction between CrpF46 and PCNA in HeLa cells by co-immunoprecipitation (Co-IP) experiment. We also found their subcellular co-localization in HeLa cytoplasm by indirect immunofluorescent experiment. To investigate the domain of CrpF46 binding with PCNA, we devided full length of CrpF46 into three fragments called CrpF46-S1, CrpF46-S2 and CrpF46-S3 respectively corresponding to the three coiled-coil domains of CrpF46, then used Pull-Down technique and discovered that CrpF46-S1 may respond to bind PCNA. At the same time, we also found that CrpF46can form complexes with PCNA binding proteins cyclin A, cyclin E, p21 and p53 in vitro. So we predicted that CrpF46 may participate in the pathway in which PCNA functions cell cycle controlling. In order to recover the functions of CrpF46 in cell cycle control, we intend to study the relationship between CrpF46 and p21 and Cdks.The interaction timing of CrpF46 and PCNA in cell cycle is also a big problem which needs to be resolved.