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 factorrelated apoptosis-induced ligand (TRAIL) signaling pathway and the ubiquitin proteasome degradation pathway. Anaplastic lymphoma kinase (ALK)1 -positive anaplastic large cell lymphoma (ALK ϩ ALCL) is a subtype of T/null cell non-Hodgkin lymphoma that is characterized by its consistent expression of CD30 and anaplastic cytology (1). ALK ϩ ALCL is relatively uncommon among adults, accounting for ϳ3% of adult non-Hodgkin lymphoma, whereas it is frequent in children, representing 10 -20% of all lymphoma (2). Approximately 80% of ALK ϩ ALCL tumors carry the t(2;5)(p23;q35) chromosomal translocation that fuses the tyrosine kinase doFrom the ‡Department

HuR-Mediated Control of C/EBPβ mRNA Stability and Translation in ALK-Positive Anaplastic Large Cell Lymphomas

“…Thus, phosphorylation of HuR by the serine-threonine kinases cdk1, PKCd, or pKCa has been reported to induce HuR translocation into the cytoplasm and modulate its RNA-binding properties (26). Since HuR is present in the cytoplasm of ALCL-derived cells, such modifications could take place in ALCLs where different serine-threonine kinases are at work (29,30), but again, they would not rely specifically on the expression of NPM-ALK. The specificity brought about by the oncogenic tyrosine kinase NPM-ALK is reflected by enhanced binding of HuR to its target mRNAs and increased engagement of HuR into actively translating polysomes.…”

The pathobiology of the oncogenic tyrosine kinase NPM-ALK: a brief update

“…For instance, we found evidence of tyrosine phosphorylation of multiple proteins in the tumor necrosis factor (TNF) signaling pathway. Using western blotting, we validated that the induction of the tyrosine phosphorylation of several TNF pathway signaling molecules by NPM-ALK, including RIP, TRAP1 and FAF1 [Wu et al 2010]. In support of their functional importance in this type of lymphoma, we knocked down the expression of TRAP1 using siRNA, and found that this treatment significantly sensitized ALK+ALCL cell lines to TRAIL-and doxorubicin-induced apoptosis.…”

“…Specifically, 506 phosphoproteins (617 phosphopeptides and 767 phosphorylation sites) were identified. Our method was designed to purify all phosphopeptides, and thus, we were able to detect a large number of serine/threonine phosphorylated proteins [Wu et al 2010].…”

“…To that end, mass spectrometry studies were performed by several laboratories, including ours, focusing on the phosphoproteomic changes induced by NPM-ALK [Rush et al 2005;Boccalatte et al 2009;Wu et al 2010]. In order to optimize the sensitivity and specificity of our assay, we employed an approach combining sequential affinity purification of phosphopeptides and liquid chromatography-tandem mass spectrometry.…”

Nucleophosmin-anaplastic lymphoma kinase: the ultimate oncogene and therapeutic target

“…Another phosphoproteomic analysis of cells with NPM-ALK expression identified targets in TNF family-related apoptotic and metabolic pathways. 66 Proteomic analysis of Jurkat cells expressing NPM-ALK identified increased expression of proteins involved in signaling pathways downstream of NPM-ALK, including STAT3. 67 A different proteomic screen identified 254 NPM-ALK binding partners involved in wide-ranging biological processes, including metabolic pathways.…”