Abstract
Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.
Highlights
Nucleoside diphosphate kinases (NDPKs) are ubiquitous enzymes which catalyze the transfer of the terminal phosphate group from (d)NTPs to (d)NDPs through a high-energy phosphohistidine intermedier [1]
We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation
At least 80% of the cytoplasmic NDPK activity is being exerted by NME1/NDPK A and NME2/NDPK B, which combine to form a series of homoor heterohexameric isoenzymes (A6, A5B1, B6) [4]
Summary
Nucleoside diphosphate kinases (NDPKs) are ubiquitous enzymes which catalyze the transfer of the terminal phosphate group from (d)NTPs to (d)NDPs through a high-energy phosphohistidine intermedier [1]. These enzymes are considered to be key players in the maintenance of the cellular NTP pool. NME1 and NME2 separated through cis-duplication from a common ancestral gene after the emergence of amphibians and are considered to be paralogs [6] Both NME1 and NME2 possess the NDP kinase active site motif (NXXHG/ASD) and are enzymatically active with similar kinetic parameters [7]. In spite of intense research in this area, we still do not have a mechanistic model of metastasis suppression activity of NME proteins
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