Identification of β-Subunit of GTP-Binding Regulatory Protein in Mitotic Spindle

BACKGROUND: The heterotrimeric GTP-binding regulatory protein (G protein) is important in membrane signal transduction. Since the function of theβ-subunit of G protein (Gβ) in tumor cells is not well-documented, identification of G, in tumor cells was performed. EXPERIMENTAL DESIGN: Immunolocalization, Western blotting, and immunoprecipitation of Gβ, in mammalian tumor and normal cells were investigated using rabbit antisera against amino and carboxyl terminal peptide fragments of Gβ. A human nasopharyngeal carcinoma cell line (NPC-TW039) was used as the cell model because of its short doubling time. RESULTS: Anti-Gβ immunoreactivity was found to be associated with the plasma membrane and the mitotic spindle throughout the mitotic phase of cell replication. Colcemid pretreatment resulted in random distribution of the anti-G4 reaction product in the mitotic cells. The same phenomenon was also seen in various other tumor and normal cell lines. When solubilized membranous, cytosolic, and mitotic spindle fractions were analyzed by Western blotting, Gβ (35 ± 1 kDa) was found to be associated with the plasma membrane and mitotic spindle fractions. Immunoprecipitation of isolated mitotic spindles with anti-Gβ further confirmed these results. CONCLUSIONS: These findings indicate that Gβ, is closely associated with the mitotic spindle as well as the plasma membrane and may be important in regulation of cell mitosis in addition to transmembrane signal transduction.