Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C-gamma to mediate its mitogenicity

Mol Cell Biol. 1998 Dec;18(12):6951-61. doi: 10.1128/MCB.18.12.6951.

Abstract

Large-cell anaplastic lymphoma is a subtype of non-Hodgkin's lymphoma characterized by the expression of CD30. More than half of these lymphomas have a chromosomal translocation, t(2;5), that leads to the expression of a hybrid protein comprised of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK). Here we show that transfection of the constitutively active tyrosine kinase NPM-ALK into Ba/F3 and Rat-1 cells leads to a transformed phenotype. Oncogenic tyrosine kinases transform cells by activating the mitogenic signal transduction pathways, e.g., by binding and activating SH2-containing signaling molecules. We found that NPM-ALK binds most specifically to the SH2 domains of phospholipase C-gamma (PLC-gamma) in vitro. Furthermore, we showed complex formation of NPM-ALK and PLC-gamma in vivo by coimmunoprecipitation experiments in large-cell anaplastic lymphoma cells. This complex formation leads to the tyrosine phosphorylation and activation of PLC-gamma, which can be corroborated by enhanced production of inositol phosphates (IPs) in NPM-ALK-expressing cells. By phosphopeptide competition experiments, we were able to identify the tyrosine residue on NPM-ALK responsible for interaction with PLC-gamma as Y664. Using site-directed mutagenesis, we constructed a comprehensive panel of tyrosine-to-phenylalanine NPM-ALK mutants, including NPM-ALK(Y664F). NPM-ALK(Y664F), when transfected into Ba/F3 cells, no longer forms complexes with PLC-gamma or leads to PLC-gamma phosphorylation and activation, as confirmed by low IP levels in these cells. Most interestingly, Ba/F3 and Rat-1 cells expressing NPM-ALK(Y664F) also show a biological phenotype in that they are not stably transformed. Overexpression of PLC-gamma can partially rescue the proliferative response of Ba/F3 cells to the NPM-ALK(Y664F) mutant. Thus, PLC-gamma is an important downstream target of NPM-ALK that contributes to its mitogenic activity and is likely to be important in the molecular pathogenesis of large-cell anaplastic lymphomas.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites / physiology
  • Cell Line
  • Enzyme Activation / physiology
  • Inositol Phosphates / metabolism
  • Isoenzymes / metabolism*
  • Lymphoma, Large B-Cell, Diffuse / enzymology*
  • Mitogens / physiology*
  • Mutagenesis, Site-Directed / genetics
  • Nuclear Proteins / metabolism*
  • Peptide Mapping
  • Phospholipase C gamma
  • Phosphopeptides / chemistry
  • Phosphorylation
  • Protein Binding / physiology
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Rats
  • Signal Transduction / physiology
  • Transfection / genetics
  • Type C Phospholipases / metabolism*
  • src Homology Domains / physiology

Substances

  • Inositol Phosphates
  • Isoenzymes
  • Mitogens
  • Nuclear Proteins
  • Phosphopeptides
  • nucleophosmin
  • p80(NPM-ALK) protein
  • Protein-Tyrosine Kinases
  • Type C Phospholipases
  • Phospholipase C gamma