CrpF46是本实验室发现的一种中心体蛋白，前期研究发现它的功能涉及细胞的增殖、运动等方面。结合现有的研究水平和磷酸化位点预测软件分析，我们推测磷酸化修饰调控可能是其在细胞中发挥功能的一种重要的修饰调节方式。通过32P放射自显影实验，我们发现CrpF46确实能够发生磷酸化修饰，但是反应强度相比阳性对照hCDC25C弱很多，提示其磷酸化位点很少。此外，CrpF46的磷酸化修饰具有细胞周期性，即在有丝分裂期发生磷酸化，而在间期不发生磷酸化修饰。 CrpF46的定位同样具有周期性，其间期只定位于中心体上，有丝分裂期则弥散的分布于整个胞质。通过截断突变CrpF46蛋白，研究其确切的磷酸化修饰位点，发现其磷酸化位点存在于437~480位氨基酸残基中，并且这一区域与其在中心体定位的必需区域420~454位氨基酸残基有一段重合；重合区域包含了潜在的苏氨酸磷酸化位点，表明其磷酸化可能影响其在中心体上的精确定位。根据前期发现的CrpF46和中心体功能相关的主要激酶PLK1及中心体标志性蛋白γ-tubulin能发生免疫共沉淀反应，并且敲除CrpF46的细胞株阻断于细胞S期的实验结果，我们通过体外磷酸化实验发现，PLK1能够磷酸化CrpF46蛋白，这显示在有丝分裂期，CrpF46发生磷酸化的激酶可能就是PLK1，并以此激活CrpF46在有丝分裂期的功能。然而，当突变了437~480位氨基酸残基中潜在的PLK1磷酸化位点后，发现其仍然可以磷酸化，提示我们CrpF46存在不止一个磷酸化位点，在437~530位氨基酸中可以存在多个磷酸化位点。对CrpF46磷酸化修饰调控的研究，有利于进一步了解细胞分裂中中心体组装、复制等的作用机理。

A centrosomal protein CrpF46 was identified by our group. The preliminary work showed its function in the cell involved in the proliferation, mobility and so on. According to current researching level and the analysis of phosphorylation site prediction software, we inferred that the phosphorylation modification regulation is likely to play a significant role in its cellular function. And than, by the 32P autoradiography experiments, we found that CrpF46 indeed was phosphorylated in vitro. But the intension of CrpF46 was very weaker than that of the positive control, human CDC25C protein. This implied that CrpF46 had less phosphorylation site. In addition, the phosphorylation modification of CrpF46 was of periodicity, namely, phosphorylated in the mitosis and unphosphorylated in the inerphase. CrpF46 location was also of periodicity, which located exclusively to the centrosome during interphase, and dispersed throughout the cytoplasm at the onset of mitosis. In order to research its precise phosphorylation sites, we broke CrpF46 into some fragments and found that its phosphorylation sites exist in the 437~480 amino acids fragment. Coincidently, there is a length of overlapping between this region and another region which is necessary for CrpF46 location on the centrosome and this length of overlapping contains some potential threonine phosphorylation sites. All the above implied that CrpF46 phosphorylation is likely to influence its precise location on the centrosome. Based on the preliminary work that CrpF46 can co-immunoprecipitated with γ-tubulin, a marker protein of centrosome and PLK1, a primary centrosomal function related protein kinase, and the knockdown CrpF46 cell line would be arrested in the S phase, we proved that PLK1 can phosphorylate CrpF46 in vitro, which showed that CrpF46 phosphorylation kinase is PLK1 and PLK1 activated CrpF46 function in the mitosis. However, mutating the potential phosphorylation sites corresponding to PLK1 on the 437~480 amino acids region of CrpF46, we discovered CrpF46 can still be phosphorylated. This implied that CrpF46 phosphorylation site is not typical one corresponding to PLK1, or CrpF46 has more than one phosphoryaltion site.The study of CrpF46 phosphorylation will be conducive to further understanding the mechanism of the function of centrosomal assembly and dupilation in cell division.