Nucleoside diphosphate kinase beta (Nm23-R1/NDPK beta) is associated with intermediate filaments and becomes upregulated upon cAMP-induced differentiation of rat C6 glioma

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell p roliferation, differentiation, development, tumor progression, and metastas is. Nevertheless, no consensus exists about the molecular mechanism by whic h Nm23/NDPK isoforms exert their role in these processes. We investigated t he expression of the rat Nm23-R1/NDPK beta and Nm23-R2/NDPK alpha isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respec tively, upon cAMP-induced differentiation of rat C6 glioma cells and demons trated a differential interaction with intermediate filaments. Semiquantita tive RT-PCR, immunoblotting, and flow cytometry showed a constitutive expre ssion of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upreg ulation of the Nm23-R1 mRNA and protein, respectively, was observed, In con trast, the expression of Nm23-R2 remained unchanged. Localization of both i soforms with confocal laser scanning microscopy demonstrated a punctate ret icular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimen tin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a p ossible function of Nm23 isoforms in cell proliferation, differentiation, a nd tumor-associated mechanisms is discussed. (C) 2000 Academic Press.