Identification of multiple SNT-binding sites on NPM-ALK oncoprotein and their involvement in cell transformation.
The t(2;5) chromosomal translocation occurs in anaplastic large-cell lymphoma arising from activated T lymphocytes. This genomic rearrangement generates the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) oncoprotein that is a chimeric protein consisting of parts of the nuclear protein NPM and ALK receptor protein-tyrosine kinase. We used yeast two-hybrid screening to identify an adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT)-2 as a new partner that interacted with the cytoplasmic domain of ALK. Immunoprecipitation assay revealed that SNT-1 and SNT-2 interacted with NPM-ALK and kinase-negative NPM-ALK mutant. Y156, Y567 and a 19-amino-acid sequence (aa 631-649) of NPM-ALK were essential for this interaction. The interaction through Y156 and Y567 was dependent on phosphorylation of these tyrosines, whereas the interaction through the 19-amino-acid sequence was independent of phosphorylation. NPM-ALK mutant protein mutated at these three binding sites showed significantly reduced transforming activity. This transformation-defective NPM-ALK mutant still interacted with signal transducing proteins such as phospholipase C-gamma and phosphatidylinositol 3-kinase, which were previously reported to be relevant to NPM-ALK-dependent tumorigenesis. These observations indicate that the three SNT-binding sites of NPM-ALK are important for its transforming activity. This raises a possibility that SNT family proteins play significant roles in cellular transformation triggered by NPM-ALK, which though remains to be verified.
Chikamori
M
M
Division of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.
Fujimoto
J
J
Tokai-Nishizumi
N
N
Yamamoto
T
T
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D025941:Protein Interaction Mapping; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; C488187:FRS2alpha protein, mouse; C485162:Frs3 protein, mouse; D008565:Membrane Proteins; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases