MECHANISMS OF RAF ACTIVATION

Raf-1蛋白丝氨酸/苏氨酸激酶作为一种重要的穿梭蛋白 连接生长因子受体和蛋白质刺激的酶 激酶C在膜上与早期生长反应基因的激活 在细胞核中。 最近，我们发现英国皇家空军的另一名成员 B-Raf家族在神经生长因子（NGF） 刺激. Raf-1和B-Raf激活的受体偶联是 p21 Ras的作用。 细胞的生长因子刺激导致 Raf-1的磷酸化和伴随的激酶活化增加， 拉夫 最近，一个负调控磷酸化的Raf-1， 已描述了A-激酶。 我们已经研究了cAMP对 Ras-Raf丝裂原活化蛋白激酶（MAPK）信号级联和 PC 12细胞的神经元分化。 我们研究了 cAMP对Raf-1和B-Raf的活化的影响，其也在 PC 12细胞。 在大鼠成纤维细胞中，Raf-1的激活强烈地抑制了成纤维细胞的生长。 被cAMP抑制。 与此相反，cAMP不抑制NGF或表皮生长因子， 生长因子（EGF）诱导的B-Raf活化（通过 MAP/Erk激酶-1（MEK 1）的磷酸化。 由于B-Raf缺少PKA 磷酸化位点，这与Raf-1的cAMP抑制一致 由PKA磷酸化介导。 B-Raf的MEK激酶活性 NGF或EGF诱导的细胞凋亡至少是Raf-1的10倍，这表明 B-Raf主要负责MAPK的激活。 因为B-Raf 激活未被阻断，cAMP不抑制Ras-Raf-MAPK级联反应 在PC 12细胞中。 相比之下，cAMP可以与Ras-Raf途径协同作用， 来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.