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Blood, 1 October 2007, Vol. 110, No. 7, pp. 2600-2609.
Prepublished online as a Blood First Edition Paper on May 30, 2007; DOI 10.1182/blood-2006-01-028647.
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Submitted January 17, 2006
Accepted May 17, 2007
NPM/ALK binds and phosphorylates the RNA/DNA binding
protein PSF in anaplastic large cell lymphoma
Annamaria Galietta*, Rosalind H Gunby, Sara Redaelli, Paola Stano, Cristiana Carniti, Angela Bachi, Philip W Tucker, Carmen J Tartari, Ching-Jung Huang, Emanuela Colombo, Karen Pulford, Miriam Puttini, Rocco G Piazza, Holger Ruchatz, Antonello Villa, Arianna Donella-Deana, Oriano Marin, Danilo Perrotti, and Carlo Gambacorti-Passerini
Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy
Department of Experimental Oncology, The National Cancer Institute of Milan, Milan, Italy
DIBIT, San Raffaele Scientific Institute, Milan, Italy
Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX, United States
Department of Experimental Oncology, The European Institute of Oncology, Milan, Italy
Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford, United Kingdom
Microscopy and Image Analysis Consortium, Dept of Neuroscience, University of Milano-Bicocca, Monza, Italy
Department of Biological Chemistry, Institute of Neuroscience of the Italian National Research Council, University of Padua, Padua, Italy
Dept. Microbiology, Immunology, Virology, & Medical Genetics, Human Cancer Genetics Program & Ohio State University Comprehensive Cancer Center, Columbus, OH, United States
Department of Oncology, McGill University, Montreal, Canada
* Corresponding author; email: galietta_am{at}yahoo.it.
The oncogenic fusion tyrosine kinase, NPM/ALK, induces cellular transformation in anaplastic large cell lymphomas (ALCL) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA binding proteins were found to co-immunoprecipitate with NPM/ALK, including the multi-functional polypyrimidine tract binding protein-associated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine phosphorylated in NPM/ALK expressing cell lines and in primary ALK+ ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK+ cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK transformed cells.
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