ROLE OF THE NUCLEOPHOSMIN (NPM) PORTION OF THE NON-HODGKINS LYMPHOMA-ASSOCIATED NPM-ANAPLASTIC LYMPHOMA KINASE FUSION PROTEIN IN ONCOGENESIS

The NPM-ALK fusion gene, formed by the t(2;5)(p23;q35) translocation i n non-Hodgkin's lymphoma, encodes a 75-kDa hybrid protein that contain s the amino-terminal 117 amino acid residues of the nucleolar phosphop rotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase ALK (anaplastic lymphoma kinase), Here, we demonstrate the transforming ability of NPM-ALK and show that oncog enesis by the chimeric protein requires the activation of its kinase f unction as a result of oligomerization mediated by the NPM segment, Se dimentation gradient experiments revealed that NPM-ALK forms in vivo m ultimeric complexes of approximately 200 kDa or greater that also cont ain normal NPM. Cell fractionation studies of the t(2;5) translocation -containing lymphoma cell line SUP-M2 showed NPM-ALK to be localized w ithin both the cytoplasmic and nuclear compartments, Immunostaining pe rformed with both polyclonal and monoclonal anti-ALK antibodies confir med the dual location of the oncoprotein and also indicated that NPM-A LK is abundant within both the nucleoplasm and the nucleolus, An intac t NPM segment is absolutely required for NPM-ALK-mediated oncogenesis, as indicated by our observation that three different NPM-ALK mutant p roteins lacking nonoverlapping portions of the NPM segment were each u nable to form complexes, lacked kinase activity in vivo, and failed to transform cells, However, NPM could be functionally replaced in the f usion protein with the portion of the unrelated translocated promoter region (TPR) protein that activates the TPR-MET fusion kinase by media ting dimerization through its leucine zipper motif, This engineered TP R-ALK hybrid protein, which transformed cells almost as efficiently as NPM-ALK, was localized solely within the cytoplasm of cells, These da ta indicate that the nuclear and nucleolar localization of NPM-ALK, wh ich probably occur because of transport via the shuttling activity of NPM, is not required for oncogenesis. Further, the activation of the t runcated ALK protein by a completely heterologous oligomerization doma in suggests that the functionally important role of the NPM segment of NPM-ALK in transformation is restricted to the formation of kinase-ac tive oligomers and does not involve the alteration of normal NPM funct ions.