Pim1是一种丝氨酸/苏氨酸激酶，在多种肿瘤细胞中高表达，呈现出癌基因特性。最近本课题组的研究却发现：癌基因Pim1可调节成纤维细胞的衰老进程，过表达Pim1可诱导细胞衰老，而抑制Pim1表达可延缓细胞衰老进程。关于Pim1调节细胞衰老的分子机制目前仍不清楚。为此，本项目通过蛋白质免疫沉淀和质谱技术，筛选出了与Pim1相互作用的下游靶蛋白，我们试图通过对靶蛋白的研究来揭示Pim1调节细胞衰老的信号通路，本课题选择其中一种靶蛋白-UHRF1进行了重点研究。前期研究结果表明：Pim1可直接结合并磷酸化UHRF1。那么，UHRF1磷酸化后活性有何变化，该变化怎样影响 Pim1调节的细胞衰老过程，这正是本课题的研究重点。本课题是在我们以前研究工作上的扩展和深入，其研究成果将不仅对细胞衰老机制的研究注入新的内容，而且对相关肿瘤发生机制的探讨提供新线索。

Pim1 is a proto-oncogene,encoding a serine/threonine kinase Pim1 that regulates cell proliferation and growth. Overexpression of Pim1 leads to tumor formation in mice, while complete Pim1 knockout has no observable phenotype. Pim1 expression in normal tissues is nearly undetectable. However, in hematopoietic malignancies and in many solid tumors, increased Pim1 expression has been shown to correlate with the stage of disease.Our recent research revealed ectopic expression of Pim1 resulted in premature senescence, inhibition of Pim1 alleviated both replicative and oncogene-induced senescence.These results demonstrated that Pim1 acts as a positive regulator of cellular senescence, which implies that Pim1 likely has characteristics of tumor suppressor. Nevertheless, the molecular mechanisms of Pim1 regulating cellular senescence remains unclear. For the sake of revealing the Pim1 signaling pathway in regulating cellular senescence,we screened the downstream target proteins of Pim1 in senescent 2BS and IMR90 cells in which the senescence is induced by Pim1 overexpression,through performing immunoprecipitation, silver stain and mass spectrometry, and selected UHRF1 to carry out further research .UHRF1 is a 90KDa nuclear protein with multiple domains. Previous studies have proposed that UHRFl plays an important role in DNA methylation, heterochromatin formation and gene expression.To test whether Pim1 directly interacts with UHRFl, we performed coimmunoprecipitation and in vitro GST pull-down assays.The results confirmed direct binding of Pim1 and UHRFl in vitro.To determine whether direct interaction between Pim1 and UHRFl results in phosphorylation of UHRFl by Pim1, immunoprecipitation and in vitro kinase assay were performed, the results confirmed that UHRFl can be phosphorylated by Pim1.We then utilized mass spectrometry to find out the UHRFl phosphorylation sites and preliminarily screened 4 phospho-sites on S311、S401、S664 and T690.Our following work is aimed at the study of the phosphorylation of UHRFl whether leads to the change of its stability and biological activity, try to reveal the effect of Pim1 on the function of UHRF1, and UHRF1's functional changes whether play an important role in cellular senescence. By exploring the mechanism of UHRF1’s function on Pim1 induced cellular senescence, we try to provide new clues for understanding of cellular senescence.

Pim1是一个原癌基因，它编码一种丝氨酸/苏氨酸激酶，可以调节细胞增殖、生存、分化和凋亡。有研究报导过表达Pim1能诱导细胞衰老。然而，其分子机制仍不清楚。我们研究发现UHRF1是Pim1的底物，Pim1能够在311位点磷酸化UHRF1，进而导致UHRF1的降解。UHRF1的降解导致基因组DNA低甲基化，而基因组DNA低甲基化又导致基因组不稳定。UHRF1的降解也可增加p16的表达，进而导致细胞衰老。免疫组化结果显示在癌前病变组织中UHRF1和Pim1的表达量相关。综上所述，下调UHRF1是Pim1诱导细胞衰老的一种机制。