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Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells.

ABSTRACT: Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin-ALK and echinoderm microtubule-associated protein-like 4-ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.

SUBMITTER: Sattu K

PROVIDER: S-EPMC3892176 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells.

Sattu Kamaraj K Hochgräfe Falko F Wu Jianmin J Umapathy Ganesh G Schönherr Christina C Ruuth Kristina K Chand Damini D Witek Barbara B Fuchs James J Li Pui-Kai PK Hugosson Fredrik F Daly Roger J RJ Palmer Ruth H RH Hallberg Bengt B

The FEBS journal 20130822 21

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin-ALK and echinoderm microtubule-associated protein-like 4-ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and b ...[more]

PMID: 23889739

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Project description: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.

Dataset Information

Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells.

Publications

Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells.

Similar Datasets

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