Proteomic Phosphosite Analysis Identified Crucial NPM-ALK-Mediated NIPA Serine and Threonine Residues

Int J Mol Sci. 2019 Aug 20;20(16):4060. doi: 10.3390/ijms20164060.


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.

Keywords: ALCL; NIPA (ZC3HC1); NPM-ALK; phosphoproteomics.

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Line
  • Flow Cytometry
  • Humans
  • Immunoprecipitation
  • Lymphoma, Large-Cell, Anaplastic / metabolism
  • Microscopy, Fluorescence
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Proteomics / methods*
  • Serine / chemistry*
  • Signal Transduction
  • Threonine / chemistry*


  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • Nuclear Proteins
  • Phosphoproteins
  • ZC3HC1 protein, human
  • Threonine
  • Serine