THE GTP-BINDING PROTEIN G(I-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 G(i) is present in Balb/c3T3 cells. Therefore, G(i) was implicated as important in the signal transduction of EGF and insulin receptors leading to mitosis. Our previous studies of the role of G(i) in cell division have shown that the alpha-subunit of G(i)(G(i alpha)) is induced to translocate from the cell periphery to the nucleus by these growth factors, and in the nucleus of dividing cells G(i alpha) binds to the separating chromatin. As protein phosphorylations are essential components of the messenger systems from these receptors, we have examined whether G(i) could be functionally coupled to protein kinases in the activated cell. We have found that G(i alpha 2) is directly linked to a serine kinase in Balb/c3T3 fibroblasts, and that G(i alpha 2) itself is a substrate for phosphorylation in vitro. This phosphorylation of G(i 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 G(i alpha 2) could represent either a negative feedback mechanism of signal transduction, or a GTP-independent pathway of G-protein signal transduction in fibroblasts.