Fujimoto J, Shiota M, Iwahara T, Seki N, Satoh H, Mori S, Yamamoto T
Department of Oncology, University of Tokyo, Japan.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4181-6. doi: 10.1073/pnas.93.9.4181.
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 NIH 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 NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc 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 Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.
我们通过分子克隆得到了一个cDNA，它编码一种在含有染色体易位t(2;5)的Ki-1淋巴瘤细胞中独特表达且酪氨酸残基高度磷酸化的蛋白质。编码的蛋白质p80被证明是由一种蛋白质酪氨酸激酶与一种核仁蛋白B23/核磷蛋白（NPM）融合产生的。该cDNA的编码序列与先前从同一易位断点处的基因转录本中克隆的NPM-间变性淋巴瘤激酶（ALK）融合cDNA几乎相同。p80在NIH 3T3细胞中的过表达诱导了肿瘤转化，这表明p80激酶被异常激活。根据其与胰岛素受体激酶家族的序列相似性，预测p80的正常形式是一种受体型酪氨酸激酶。然而，使用COS细胞进行的免疫荧光研究表明p80定位于细胞质中。因此，酪氨酸激酶可能因其结构改变而发生亚细胞易位和激活，从而导致恶性转化。我们还表明，缺乏NPM部分的突变型p80无法转化NIH 3T3细胞。因此，NPM序列对于转化活性至关重要，这表明染色体易位与肿瘤发生有关。最后，在p80转化的细胞中，Shc和胰岛素受体底物1（IRS-1）发生酪氨酸磷酸化并与p80结合。然而，对Shc和胰岛素受体底物1的结合和磷酸化有缺陷的p80突变体能够转化NIH 3T3细胞。仍观察到这些突变体与GRB2的结合，这表明p80与GRB2的相互作用而非与Shc或IRS-1的相互作用与细胞转化有关。
