Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines: specific nuclear localization of the B subunit.

The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), encoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homo- or heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific functions. The nm23-H1 gene is implicated in tumor progression and metastasis, and the nm23-H2 gene product is a transcription factor for c-myc. To determine if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluorescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B was shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates.

A new human nm23 homologue (nm23-H5) specifically expressed in testis germinal cells.

We have identified a cDNA encoding a 212 amino acid protein (Nm23-H5) with 27-31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family. The nm23-H5 gene is located on chromosome 5q23-31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes. Nm23-H5 possesses most of the residues conserved among NDP kinases plus an additional COOH-terminus end of 55 amino acids unique to this protein. However, under usual assay conditions, Nm23-H5 expressed in Escherichia coli did not exhibit NDP kinase activity. These results suggest that Nm23-H5 is specifically involved in early stages of spermatogenesis.