NDP Kinase A參與c-myc 轉錄調控

Abstract

Nucleoside diphosphate kinase (NDPK) is an ubiquitous enzyme, which catalyzes the transfer of the terminal phosphate from a nucleoside triphosphate to a nucleoside diphosphate through a ping-pong mechanism. At present, eight members of the human NDPK family have been identified. The most widely studied members are NDPK-A and NDPK-B which are encoded by NM23-H1 and NM23-H2 genes. Accumulating evidence indicates that NDPK-A, but not NDPK-B, is strongly correlated with tumor metastasis even though the two proteins share 88% identical amino acid residues. NDPK-B was identified as PuF, which is a transcriptional regulator of c-myc that binds to the nuclease-hypersensitive element III1 (NHE) III1 in the c-myc promoter. Our laboratory previously found that NDPK-A also specifically binds c-myc NHEIII1 in vitro and regulates the c-myc promoter activity in vivo. Compared to NDPK-B, however, NDPK-A displayed a significantly lower binding affinity toward c-myc DNA based on EMSA and UV-crosslinking. In my study, I have examined the individual and collaborative effects of NDPK-A and NDPK-B on c-myc transcriptional regulation in NB69 neuroblastoma cells and cervical cancer HeLa cells. Using the CAT assay, I found that the phosphotransferase activity or intra-molecular disulfide bond of NDPK-A was not essential for its regulation of c-myc transcription. Ectopic expression of wide-type NDPK-A (NDPKAWT) and metastasis-associated S120G mutation (NDPKAS120G), negatively regulated c-myc mediated transcription in a dose-dependent manner. Similarity, NDPK-B also displayed an inhibitory effect on c-myc transcription in both cell lines. When combined with NDPK-B, NDPK-A suppressed c-myc transcription more strongly than either protein alone. On the other hand, knockdown of endogenous NDPK-A and/or NDPK-B enhanced c-myc transcription. Under oxidative stress, knockdown of NDPK-A decreased cell viability by ~15% relative to the control cells. Knockdown of both NDPK-A and NDPK-B further decreased cell viability by ~50% after H2O2 treatment. Additionally, knockdown of NDPK-A increased the migration potential, whereas knockdown of NDPK-B reduced its migration capacity compared with the control. However, knockdown NDPK-A and/or NDPK-B did not significantly affect the proliferation of HeLa cells. These findings provide a link between metastasis-associated alterations of NDPK-A and c-myc deregulation.