Regulation of mitogenic signaling pathways which involve the function of GTP-binding protein-Possible involvement of protein tyrosine phosphorylation.

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We have examined the possible invements of pertussis toxin toxin(PT)-sensitive guanosine triphosphate(GTP)-binding protein(Gp)and protein kinase C(PKC)in the mitogenic signaling pathways of various growth factors by using PT-pretreated and/or 12-0-tetradecanoyl phorbol-13-acetate(TPA)-pretreated mouse fibroblasts. Effects of PT-pretreatment(inactivation of PT-sensitive Gp)and TPA pretreatment(depletion of PKC)on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors : mitogenic response of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells ; responses to epidermal growth factor(EGF), platelet-derived growth factor(PDGF)and vanadate were reduced to -50% both in PT- and TPA-pretreated cells compared with native cells ; response to basic fibroblast growth factor(bFGF)was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus growth factors … More examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway.Mitogenic responses not only to PDGF, EGF and BFGF, but also to vanadate were attenuated in PKC-depleted cells, indicating that signal transduction pathways of these mitogens involves the function of PKC. Receptor molecules for EGF, PDGF and bFGF have been reported to possess tyrosine kinase activity, upon which mitogenic signal transduction of EGF, PDGF and BFGF totally depend. Vanadate is a potent inhibitor of phosphotyrosyl protein phosphatase. it thus seems very likely that the levels of tyrosine phosphorylation are increased in those mitogen-stimulated cells. The most plausible mechanism for the link between the increased tyrosine phosphorylation and the activation of PKC is that the activated receptor tyrosine kinases induce, either directly or indirectly, the phosphorylation of some elements that are involved in the PKC-activating pathway. In this regard, PDGF-, EGF- and vanadate-induced DNA synthesis, not only in PKC-depleted cells but also in PT-sensitive Gp-inactivated cells, is reduced to -50% compared with native cells. These results thus suggest that PDGF and EGF stimulate tyrosine phosphorylation of the component whose function lies upstream of PT-sensitive Gp on their mitogenic signaling pathways. In accordance with this possibility, PDGF- and EGF-stimulated tyrosine phosphorylation of p2l ras_GTPase-activating protein(GAP)has been reported very recently. The physiological significance, as well as the precise mechanism, of the mitogen-stimulated tyrosine phosphorylation of GAP is currently being investigated. Less