The association of the Nm23-M1 protein and beta-tubulin correlates with cell differentiation.
The Nm23 protein is a nucleoside diphosphate kinase (NDPK) and is thought to play a critical role in metastatic behavior. It has been reported that a NDPK activity is present in microtubules assembled in vitro. Since microtubule assembly is determinant in cell growth and differentiation, we investigated whether Nm23-M1 forms molecular complexes with beta-tubulin in murine cells either actively proliferating or differentiating. For this purpose a polyclonal antibody against the GST-Nm23-M1 fusion protein was generated and employed to detect Nm23-M1/beta-tubulin complexes in murine tumor cells derived from the Lewis lung carcinoma (3LL) and in undifferentiated and differentiated myogenic cells (C2C12). Immunoblotting and immunoprecipitation experiments performed using the anti-fusion protein antibody demonstrated that the Nm23-M1 protein is detectable in in vitro tumor cell lines and in in vivo primary tumors but not in spontaneous lung metastases. These data are in good agreement with data previously reported. Immunoprecipitation experiments demonstrated that the Nm23-M1 protein forms complexes with beta-tubulin in in vitro tumor cell lines, but not in primary tumors. Furthermore, the Nm23-M1 protein forms complexes with beta-tubulin in myogenic cells prior to and after differentiation. Interestingly, however, the level of the Nm23-M1/beta-tubulin complexes is remarkably increased in differentiated myotubes. In conclusion, the results indicate that the Nm23-M1 protein forms molecular complexes with beta-tubulin and that the number of complexes increases during the differentiation process of murine cells.
Lombardi
D
D
Dipartimento di Medicina Sperimentale, Università degli Studi, L'Aquila, Italy.
Sacchi
A
A
D'Agostino
G
G
Tibursi
G
G
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D002454:Cell Differentiation; D002455:Cell Division; D017931:DNA Primers; D015151:Immunoblotting; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D018482:Muscle, Skeletal / Q000166:cytology; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism*; D014404:Tubulin / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D017931:DNA Primers; D054778:NM23 Nucleoside Diphosphate Kinases; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014404:Tubulin; C517939:Nme1 protein, mouse; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins