Molecular Mechanism Of The Two Important Tyrosine Residues Within NPM-ALK

Anaplastic large cell lymphoma (ALCL) is an aggressive type of malignant lymphoma. ALK is expressed and constitutively activated because of one of several chromosomal aberrations involving the ALK gene on chromosome2p23. The translocation t(2;5)(p23;q35), which also involves the NPM (nucleophosmin)gene, constitutes approximately85%of these aberrations. One major outcome of this translocation is the generation of the NPM-ALK chimeric protein and a constitutively active ALK. There is agreement that NPM-ALK plays a crucial role in the pathogenesis of NPM-ALK+T cell lymphoma. Previous studies have shown that NPM-ALK associates and functionally interacts with an array of downstream molecules known to regulate cell survival and growth by phosphorylation of some tyrosine residues. However, the exact mechanisms involved are not completely understood.In this study, eukaryotic expression plasmid pEGFP-N1NPM-ALK and pEGFP-N1NPM-ALKY64,664F were constructed by using subcloning technology and were transfected into human embryonic kidney293T cells. Using RT-PCR. Western blot and fluorescence microscopy, expression of GFP and NPM-ALK was confirmed.NPM-ALKY644,664F expression plasmid makes the level of phosphorylation of downstream signaling molecules is significantly reduced. Then, the recombinant plasmid pEGFP-N1NPM-ALK and pEGFP-NI NPM-ALKY644,664F was electrotransfected into Jurkat cells. GFP-positive cells was screened out by G418pressure and flow cytometry. Consistent with293T results, NPM-ALKY644,6644F expression makes the level of phosphorylation of downstream signaling molecules is significantly reduced in Jurkat cells. Meanwhile, the phosphorylations of NPM-ALK and IGF-1R were reduced. Importantly, we found that the dual mutation of Tyr644and Tyr664diminishes the oncogenic effects of NPM-ALK, including its ability to induce colony formation and cell cycle arrest at G1phase.Therefore,644and664tyrosine residues are crucial for the oncogenic potential of NPM-ALK. Our previous studies have shown that the decoction of Toona sinensis and Moschus inhibits different type of cancer cells. We examined the growth effect of the decoction on our established stable Jurkat cells. The data showed that the dual mutation of Tyr644and Tyr664increased the resistance to the decoction. The result indicated that644and664tyrosine residues could become potential target for drug screening.In short, the dual mutation of Tyr644and Tyr664abrogates the association and interactions between NPM-ALK and IGF-1R, reduces the phosphorylation of downstream signaling molecules, and diminishes the oncogenic effects of NPM-ALK. The two important tyrosine residues could be used as potential target for future drug screening.