Abstract
Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma that shows in 60% of cases a translocation t(2;5)(p23;q35), which leads to the expression of the oncogenic kinase NPM-ALK. The nuclear interaction partner of ALK (NIPA) defines an E3-SCF ligase that contributes to the timing of mitotic entry. It has been shown that co-expression of NIPA and NPM-ALK results in constitutive NIPA phosphorylation. By mass spectrometry-based proteomics we identified nine serine/threonine residues to be significantly upregulated in NIPA upon NPM-ALK expression. Generation of phospho-deficient mutants of the respective phospho-residues specified five serine/threonine residues (Ser-338, Ser-344, Ser-370, Ser-381 and Thr-387) as key phosphorylation sites involved in NPM-ALK-directed phosphorylation of NIPA. Analysis of the biological impact of NIPA phosphorylation by NPM-ALK demonstrated that the ALK-induced phosphorylation does not change the SCFNIPA-complex formation but may influence the localization of NIPA and NPM-ALK. Biochemical analyses with phospho-deficient mutants elucidated the importance of NIPA phosphorylation by NPM-ALK for the interaction of the two proteins and proliferation potential of respective cells: Silencing of the five crucial NIPA serine/threonine residues led to a highly enhanced NIPA-NPM-ALK binding capacity as well as a slightly reduced proliferation in Ba/F3 cells.
Highlights
Anaplastic large cell lymphoma (ALCL) belongs to the group of aggressive non-Hodgkin’s lymphomas (NHL) and was first described as a distinct tumor entity in 1985 [1]
To demonstrate that nuclear interaction partner of anaplastic lymphoma kinase (ALK) (NIPA) is phosphorylated by ALK expression and not by commonly upregulated pathways (i.e., MEK-pathway) in ALCL cells, we analyzed the phosphorylation of NIPA in ALK+ (DEL, TS, JB6) and ALK- (Jurkat, FEPD, Mac-1) ALCL cell lines with regard to the shift of NIPA in electrophoretic mobility
As shown in the 2-dimensional cell cycle analyses, both groups presented an equal amount of cells in the G2/M-phase (Figure 1B), indicating that NIPA phosphorylation is not caused by an altered cell cycle distribution between the ALK+ and ALKALCL cell entities
Summary
Anaplastic large cell lymphoma (ALCL) belongs to the group of aggressive non-Hodgkin’s lymphomas (NHL) and was first described as a distinct tumor entity in 1985 [1]. It is characterized by the expression of the CD30 receptor (Ki-1), an anaplastic morphology and a null-cell or T-cell phenotype [2]. About 70–80% of ALK + ALCLs show the translocation t(2; 5)(p23; q35), thereby expressing the chimeric NPM-ALK fusion protein [3]. The 80 kDa fusion protein NPM-ALK was first described in 1994. The molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK are still not fully understood
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