Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5)
Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5)
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DOI：
10.1073/pnas.93.9.4181
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发表时间：
1996-04-30
影响因子：
11.1
通讯作者：
Yamamoto, T
中科院分区：
文献类型：
--
作者：
Fujimoto, J;Shiota, M;Yamamoto, T
我们分子克隆了一个编码蛋白质的cDNAs，该蛋白质在含有染色体易位t(2；5)的Ki-1淋巴瘤细胞中的酪氨酸残基上有独特的表达和过度磷酸化。编码蛋白p80由蛋白酪氨酸激酶和核仁蛋白B23/核磷蛋白(NPM)融合而成。该基因的编码序列与先前从同一易位断裂点的转录本中克隆的NPM-间变性淋巴瘤激酶(ALK)融合基因的编码序列几乎完全相同。P80在NM 3T3细胞中的过表达诱导了肿瘤转化，提示p80激酶被异常激活。根据p80与胰岛素受体家族的序列相似性，预测p80的正常型是一种受体类型的酪氨酸激酶。然而，用COS细胞进行的免疫荧光研究表明，p80定位于细胞质。因此，酪氨酸激酶的亚细胞易位和激活可能通过其结构的改变而导致恶性转化。我们还发现，缺少NPM部分的突变体p80不能转化NM 3T3细胞。因此，NPM序列对转化活性是必不可少的，这表明染色体易位与肿瘤的发生有关。最后，在p80转化的细胞中，SHE和胰岛素受体底物1(IRS-1)被酪氨酸磷酸化并与p80结合。然而，与SHE和胰岛素受体底物1结合和磷酸化缺陷的p80突变体可以转化NM 3T3细胞。仍然观察到这些突变体与Grb2的关联，表明p80与Grb2的相互作用而不是与SHE或IRS-1的相互作用与细胞转化有关。
We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NM 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NM 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, She and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of She and insulin receptor substrate 1 could transform NM 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with She or IRS-1 was relevant for cell transformation.