Galietta Annamaria, Gunby Rosalind H, Redaelli Sara, Stano Paola, Carniti Cristiana, Bachi Angela, Tucker Philip W, Tartari Carmen J, Huang Ching-Jung, Colombo Emanuela, Pulford Karen, Puttini Miriam, Piazza Rocco G, Ruchatz Holger, Villa Antonello, Donella-Deana Arianna, Marin Oriano, Perrotti Danilo, Gambacorti-Passerini Carlo
Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy.
Blood. 2007 Oct 1;110(7):2600-9. doi: 10.1182/blood-2006-01-028647. Epub 2007 May 30.
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.
致癌性融合酪氨酸激酶核磷蛋白/间变性淋巴瘤激酶（NPM/ALK）在携带t(2;5)染色体易位的间变性大细胞淋巴瘤（ALCL）中诱导细胞转化。涉及NPM/ALK的蛋白质-蛋白质相互作用对于下游信号通路的激活很重要。本研究旨在鉴定可能有助于其致癌转化的新型NPM/ALK结合蛋白。使用蛋白质组学方法，发现几种RNA/DNA结合蛋白与NPM/ALK共免疫沉淀，包括多功能多嘧啶序列结合蛋白相关剪接因子（PSF）。NPM/ALK与PSF之间的相互作用依赖于活跃的ALK激酶结构域，并且在表达NPM/ALK的细胞系和原发性ALK(+) ALCL样本中发现PSF被酪氨酸磷酸化。此外，PSF在体外被证明是纯化的ALK激酶结构域的直接底物，并且PSF Tyr293被鉴定为磷酸化位点。Y293F PSF未被NPM/ALK磷酸化，并且在NPM/ALK(+)细胞中未发生定位改变。ALK融合蛋白的表达诱导PSF从细胞核转移至细胞质，并且PSF的强制过表达抑制了表达NPM/ALK的细胞的增殖并诱导了细胞凋亡。PSF磷酸化还增加了其与RNA的结合，并降低了PSF介导的对GAGE6表达的抑制。这些结果将PSF鉴定为一种新型的NPM/ALK结合蛋白和底物，并表明在NPM/ALK转化的细胞中PSF功能可能受到干扰。
