The GTP-binding protein Gi alpha 2 is directly linked to and substrate of a serine kinase in Balb/c3T3 cells.
Mitosis of Balb/c3T3 cells induced by epidermal growth factor and insulin is inhibited by pertussis toxin. Pertussis toxin inactivates certain GTP-binding proteins, of which only Gi is present in Balb/c3T3 cells. Therefore, Gi was implicated as important in the signal transduction of EGF and insulin receptors leading to mitosis. Our previous studies of the role of Gi in cell division have shown that the alpha-subunit of Gi(Gi alpha) is induced to translocate from the cell periphery to the nucleus by these growth factors, and in the nucleus of dividing cells Gi alpha binds to the separating chromatin. As protein phosphorylations are essential components of the messenger systems from these receptors, we have examined whether Gi could be functionally coupled to protein kinases in the activated cell. We have found that Gi alpha 2 is directly linked to a serine kinase in Balb/c3T3 fibroblasts, and that Gi alpha 2 itself is a substrate for phosphorylation in vitro. This phosphorylation of Gi alpha 2 is inhibited if the G-protein is first activated with GTP or inactivated with GDP, suggesting that the phosphorylation may be occurring in the guanine nucleotide binding region. We present evidence that the kinase is not a protein kinase C. Such a phosphorylation of Gi alpha 2 could represent either a negative feedback mechanism of signal transduction, or a GTP-independent pathway of G-protein signal transduction in fibroblasts.
Crouch
M F
MF
Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra.
Simson
L
L
D016475:3T3 Cells / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013873:Thionucleotides / Q000494:pharmacology
D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019204:GTP-Binding Proteins