Identification of beta-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 the beta-subunit of G protein (G beta) in tumor cells is not well-documented, identification of G beta in tumor cells was performed.


METHODS
Immunolocalization, Western blotting, and immunoprecipitation of G beta in mammalian tumor and normal cells were investigated using rabbit antisera against amino and carboxyl terminal peptide fragments of G beta. A human nasopharyngeal carcinoma cell line (NPC-TW039) was used as the cell model because of its short doubling time.


RESULTS
Anti-G beta 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-G beta 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, G beta (35 +/- 1 kDa) was found to be associated with the plasma membrane and mitotic spindle fractions. Immunoprecipitation of isolated mitotic spindles with anti-G beta further confirmed these results.


CONCLUSIONS
These findings indicate that G beta 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.
Lin
C T
CT
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Wu
H C
HC
Cheng
H F
HF
Chang
J T
JT
Chang
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000502:physiology; D006801:Humans; D007124:Immunoenzyme Techniques; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D008941:Spindle Apparatus / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D019204:GTP-Binding Proteins