Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C-gamma to mediate its mitogenicity.
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.
Bai
R Y
RY
Laboratory of Leukemogenesis, Department of Internal Medicine III, Technical University of Munich, Munich, Germany.
Dieter
P
P
Peschel
C
C
Morris
S W
SW
Duyster
J
J
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D016403:Lymphoma, Large B-Cell, Diffuse / Q000201:enzymology*; D008934:Mitogens / Q000502:physiology*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D010449:Peptide Mapping; D051966:Phospholipase C gamma; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection / Q000235:genetics; D010738:Type C Phospholipases / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology
D007295:Inositol Phosphates; D007527:Isoenzymes; D008934:Mitogens; D009687:Nuclear Proteins; D010748:Phosphopeptides; D000090243:Nucleophosmin; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma