NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.
The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) 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 coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated 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.
Galietta
Annamaria
A
Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy.
Gunby
Rosalind H
RH
Redaelli
Sara
S
Stano
Paola
P
Carniti
Cristiana
C
Bachi
Angela
A
Tucker
Philip W
PW
Tartari
Carmen J
CJ
Huang
Ching-Jung
CJ
Colombo
Emanuela
E
Pulford
Karen
K
Puttini
Miriam
M
Piazza
Rocco G
RG
Ruchatz
Holger
H
Villa
Antonello
A
Donella-Deana
Arianna
A
Marin
Oriano
O
Perrotti
Danilo
D
Gambacorti-Passerini
Carlo
C
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D000072670:PTB-Associated Splicing Factor; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic / Q000235:genetics
D004268:DNA-Binding Proteins; D000072670:PTB-Associated Splicing Factor; D016601:RNA-Binding Proteins; D019000:Phosphotyrosine; D012313:RNA; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases