Nucleoside diphosphate kinase beta (Nm23-R1/NDPKbeta) 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 proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKbeta and Nm23-R2/NDPKalpha isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular 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 vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.
Roymans
D
D
Laboratory of Cellular Biochemistry, University of Antwerp, Wilrijk-Antwerpen, B-2610, Belgium.
Willems
R
R
Vissenberg
K
K
De Jonghe
C
C
Grobben
B
B
Claes
P
P
Lascu
I
I
Van Bockstaele
D
D
Verbelen
J P
JP
Van Broeckhoven
C
C
Slegers
H
H
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology*; D003600:Cytosol / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005910:Glioma; D006801:Humans; D007382:Intermediate Filaments / Q000502:physiology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D054778:NM23 Nucleoside Diphosphate Kinases; D014157:Transcription Factors; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003994:Bucladesine; D000242:Cyclic AMP; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins; D007545:Isoproterenol