Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).

Abstract

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM).The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lym- (11, 12). We established a cell line of a Ki-1 lymphoma with t(2;5) by maintaining the tumor in severe combined immunodeficiency (SCID) mice, and the cell line was termed AMS3. By examining various signal-transducing molecules and the status of tyrosine phosphorylation of cellular proteins in the AMS3 cells, we found a unique 80-kDa tyrosine-phosphorylated protein and termed it p80. We then purified p80 using anti-phosphotyrosine antibody and determined partial amino acid sequences of some of the tryptic peptides (13). Because the sequences showed similarity to Ltk (leukocyte tyrosine kinase) of the insulin receptor family, we predicted that a novel member of this family was activated and autophosphorylated in the AMS3 cells. Our present study shows the structure of the p80 protein predicted from the cDNA clone, its subcellular localization, and its transforming activity. We also examined the relevance of Shc, IRS-1, and GRB2 to the oncogenic activity of p80. MATERIALS AND METHODSCells and Antibodies. AMS3 cells were established by implanting the tissue of Ki-1 lymphoma in SCID mice as described (13). NIH 3T3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)/5% of calf serum. COS cells were cultured in DMEM/10% of fetal calf serum. Cell culture was performed at 37°C and 5% CO2. Affinity-purified rabbit polyclonal antibody against p80 protein was prepared as described (14). Anti-Shc and anti-IRS-1 polyclonal antibodies were purchased from Upstate Biotechnology. Anti-GRB2 polyclonal antibody was from

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein

“…These results imply that NPM-MLF1 activates a novel signaling pathway of apoptotic induction. Deletion of N-terminal 83 amino acids, which are required for dimerization of NPM (Fujimoto et al, 1996;Bischof et al, 1997), only partially impaired the apoptosisinducing activity of the fusion protein, while mutants lacking nuclear localization signal completely lost the ability. This can be explained by the fact that MLF1 itself has the ability to dimerize (N Yoneda-Kato, unpublished observation).…”

“…The region of NPM retained in the NPM-MLF1 fusion protein contains most of the identi®able functional domains of NPM, including a potential Cys-X 5 -His-X 4 -His metal binding motif, one of two nuclear localization signals (NLSs), and most of the acidic amino acid clusters (Yoneda-Kato et al, 1996), while NPM-ALK and NPM-RARa contain 58 less NPMderived amino acids (Morris et al, 1994;Redner et al, 1996), suggesting that functional modi®cation of MLF1 by fusion to NPM is di erent from that for ALK and RARa. All three fusion proteins, however, retain the N-terminal domain of NPM, which are required for dimerization of the protein (Fujimoto et al, 1996;Bischof et al, 1997). The ALK and RARa genes encode a tyrosine kinase and a transcription factor, respectively, whereas the function of MLF1 remains unknown because of no signi®cant homology with any previously identi®ed proteins, suggesting that NPM-MLF1 acts through a novel pathway to contribute to myeloid neoplasia.…”

Emerging findings into molecular mechanism of brain metastasis

“…The ALK fusion protein also plays a role of linker molecule which interacts with downstream molecules during signal transduction. Its specific amino acid residues respectively bind to intracytoplasmic insulin receptor substrate 1 (IRS1), v‐src sarcoma [Schmidt‐Ruppin A‐2] viral oncogene homolog [avian] (SRC) and SRC homology 2 domain‐containing (SHC), and sequentially activate Ras/ERK pathway, simultaneously activate mTOR and its downstream ribosomal protein S6 kinase (p70S6K) and S6 ribosomal protein (S6RP), in final stimulate gene transcription and promote ribosome formation 76. Besides STAT3 and extracellular signal‐regulated kinase (ERK), PI3K also took part in regulation of ALK + NSCLC survival and anti‐apoptosis.…”

Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA

“…The NPM/ ALK chimeric protein is constitutively expressed and activated through autophosphorylation (Shiota et al, 1994;Morris et al, 1997). NPM/ALK is highly oncogenic as documented both in vitro (Fujimoto et al, 1996;Bischof et al, 1997) and in vivo (Kuefer et al, 1997;Chiarle et al, 2003). NPM/ALK executes its oncogenicity by activating a number of signal transduction proteins, including signal transducer and activator of transcription 3 (STAT3) (Zhang et al, 2002;Chiarle et al, 2008;Li and Morris, 2008).…”