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

我们用百日咳毒素(PT)敏感的三磷酸鸟苷(GTP)结合蛋白(GP)和蛋白激酶C(PKC)在不同生长因子的有丝分裂信号通路中对百日咳毒素(PT)敏感的三磷酸鸟苷(GTP)结合蛋白(GP)和蛋白激酶C(PKC)进行了研究。PT预处理(PT敏感的GP失活)和TPA预处理(去除PKC)对有丝分裂原诱导的DNA合成的影响因生长因子的不同而有显著而系统的差异：PT和TPA处理的细胞对凝血酶、蛙皮素和缓激肽的促有丝分裂反应几乎完全取消；与自然细胞相比，PT和TPA处理的细胞对表皮生长因子(EGF)、血小板衍生生长因子(PDGF)和钒的反应降低到-50%；在PT处理的细胞中，对碱性成纤维细胞生长因子(BFGF)的反应没有影响，但在TPA处理的细胞中受到一定程度的抑制。因此，生长因子…关于PT敏感的GP和PKC在其信号转导通路中的作用，更多的研究被分为三组。PT和TPA对不同有丝分裂原诱导的DNA合成的抑制作用不是相加的，提示PT敏感的GP和PKC的功能依赖于相同的信号转导途径。在PKC耗竭的细胞中，促分裂反应不仅对PDGF、EGF和bFGF，而且对钒盐的反应减弱，表明这些有丝分裂原的信号转导通路涉及PKC的功能。已报道的EGF、PDGF和bFGF3种受体分子具有酪氨酸激酶活性，其有丝分裂信号转导完全依赖于酪氨酸酶活性。钒酸盐是一种有效的磷酸酪氨酰蛋白磷酸酶抑制剂。因此，在这些丝裂原刺激的细胞中，酪氨酸磷酸化水平似乎很可能增加了。酪氨酸磷酸化增加与PKC激活之间最有可能的机制是，激活的受体酪氨酸激酶直接或间接地诱导参与PKC激活途径的某些元件的磷酸化。在这方面，PDGF、EGF和钒酸诱导的DNA合成，不仅在PKC耗竭的细胞中，而且在PT敏感的GP失活的细胞中，与天然细胞相比，减少到-50%。这些结果表明，PDGF和EGF刺激其有丝分裂信号通路中PT敏感的GP上游部分的酪氨酸磷酸化。根据这种可能性，最近报道了PDGF和EGF刺激的p21 ras_GTP酶激活蛋白(GAP)的酪氨酸磷酸化。目前正在研究丝裂原刺激的GAP酪氨酸磷酸化的生理意义和确切机制。较少

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