MECHANISMS OF RAF ACTIVATION

Raf-1蛋白丝氨酸/苏氨酸激酶作为关键穿梭蛋白发挥作用 连接生长因子受体和蛋白质刺激的酶 膜上的激酶C与早期生长反应基因的激活 在原子核中。最近，我们已经证明了Raf的另一个成员 神经生长因子(NGF)诱导PC12细胞激活B-Raf家族 刺激。Raf-1和B-Raf激活的受体偶联 受p21Ras基因调控。细胞的生长因子刺激导致 Raf-1和Raf-1的磷酸化和伴随的激酶活性增加 B-Raf。最近，Raf-1的负调节磷酸化通过 已经对A-激酶进行了描述。我们已经研究了cAMP对 Ras-Raf丝裂原活化蛋白激酶(MAPK)信号转导通路 PC12细胞的神经元分化。我们调查了它的影响。 CAMP对Raf-1和B-Raf的激活，这也在 PC12细胞。与大鼠成纤维细胞一样，Raf-1的活性很强 被营地镇压。相反，cAMP不抑制NGF或表皮 生长因子(EGF)诱导的B-Raf激活(用 MAP/ERK激酶-1(MEK1)的磷酸化。由于B-Raf缺少PKA 磷酸化位点，这与cAMP对Raf-1的抑制一致 是由PKA磷酸化介导的。B-Raf的MEK激酶活性 由NGF或EGF诱导的至少是Raf-1的10倍，提示 B-Raf主要负责MAPK的激活。因为B-Raf 激活不被阻断，cAMP不抑制Ras-Raf-MAPK级联 在PC12细胞中。相反，cAMP可以与Ras-Raf途径协同作用 激活这个细胞系统中的MAPK。

Raf-1 protein serine/threonine kinase functions as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the membrane with activation of early growth response genes in the nucleus. Recently, we have shown that another member of the Raf family, B-Raf, is activated in PC12 cells after nerve growth factor (NGF) stimulation. Receptor coupling of Raf-1 and B-Raf activation is controlled by p21Ras. Growth factor stimulation of cells results in the increased phosphorylation and concomitant kinase activation of Raf-1 and B-Raf. Most recently, a negative regulatory phosphorylation of Raf-1 by A-kinase has been described. We have studied the effects of cAMP on the Ras-Raf mitogen-activated protein kinase (MAPK) signaling cascade and neuronal differentiation of PC12 cells. We investigated the effect of cAMP on activation of both Raf-1 and B-Raf, which is also expressed in PC12 cells. As in rat fibroblasts, activation of Raf-1 was strongly suppressed by cAMP. In contrast, cAMP did not inhibit NGF or epidermal growth factor (EGF)-induced activation of B-Raf (assayed by phosphorylation of Map/Erk kinase-1 (MEK1). Since B-Raf lacks a PKA phosphorylation site, this is consistent with cAMP inhibition of Raf-1 being mediated by PKA phosphorylation. The MEK kinase activity of B-Raf induced by NGF or EGF was at least tenfold greater than Raf-1, suggesting that B-Raf is primarily responsible for MAPK activation. Because B-Raf activation is not blocked, cAMP does not inhibit the Ras-Raf-MAPK cascade in PC12 cells. In contrast, cAMP can synergize with the Ras-Raf pathway to activate MAPK in this cell system.