title: Studies of phosphoproteomic changes induced by nucleophosmin-anaplastic lymphoma kinase (ALK) highlight deregulation of tumor necrosis factor (TNF)/Fas/TNF-related apoptosis-induced ligand signaling pathway in ALK-positive anaplastic large cell lymphoma.
abstract: The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found exclusively in a subset of ALK-positive anaplastic large cell lymphoma, promotes tumorigenesis by exerting its constitutively active tyrosine kinase activity. Thus, characterization of the NPM-ALK-induced changes in the phosphoproteome will likely provide insights into the biology of this oncoprotein. To achieve this goal, we used a strategy of combining sequential affinity purification of phosphopeptides and LC/MS. GP293 cells transfected with either NPM-ALK or an NPM-ALK mutant with decreased tyrosine kinase activity (negative control) were used. We identified 506 phosphoproteins detectable in NPM-ALK-expressing cells but not in the negative control. Bioinformatics analysis revealed that these phosphoproteins carry a wide diversity of biological functions, some of which have not been described in association with NPM-ALK, such as the tumor necrosis factor (TNF)/Fas/tumor necrosis factor-related apoptosis-induced ligand (TRAIL) signaling pathway and the ubiquitin proteasome degradation pathway. In particular, modulations of the TNF/Fas/TRAIL pathway by NPM-ALK were supported by our antibody microarray data. Further validation of the TNF/Fas/TRAIL pathway was performed in ALK(+) anaplastic large cell lymphoma (ALCL) cell lines with knockdown of NPM-ALK using short interference RNA, resulting in the loss of the tyrosine phosphorylation of tumor necrosis factor receptor-associated protein 1 (TRAP1) and receptor-interacting protein 1, two crucial TNF signaling molecules. Functional analyses revealed that knockdown of TRAP1 facilitated cell death induced by TRAIL or doxorubicin in ALK(+) ALCL cells. This suggests that down-regulation of TRAP1 in combination with TRAIL or doxorubicin might be a potential novel therapeutic strategy for ALK(+) ALCL. These findings demonstrated that our strategy allowed the identification of novel proteins downstream of NPM-ALK that contribute to the maintenance of neoplastic phenotype and holds great potential for future studies of cellular tyrosine kinases in normal states and diseases.
authors.affiliation: double daggerDepartment of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Fang
authors.initials: F
authors.lastname: Wu
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Jingdong
authors.initials: J
authors.lastname: Zhang
authors.forename: Leah C
authors.initials: LC
authors.lastname: Young
authors.forename: Raymond
authors.initials: R
authors.lastname: Lai
authors.forename: Liang
authors.initials: L
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D017728:Lymphoma, Large-Cell, Anaplastic / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases
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