Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).
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.
Fujimoto
J
J
Department of Oncology, University of Tokyo, Japan.
Shiota
M
M
Iwahara
T
T
Seki
N
N
Satoh
H
H
Mori
S
S
Yamamoto
T
T
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002874:Chromosome Mapping; D002889:Chromosomes, Human, Pair 2; D002895:Chromosomes, Human, Pair 5; D017931:DNA Primers; D006801:Humans; D008223:Lymphoma / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000096:biosynthesis; D000090243:Nucleophosmin; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D017931:DNA Primers; C000716967:NPM1 protein, human; D009363:Neoplasm Proteins; C000715570:Npm1 protein, mouse; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C036987:protein p80; D000090243:Nucleophosmin; D017027:Protein Tyrosine Phosphatases