THE RAF-1 SIGNALLING PATHWAY

在过去一年中，我们在确定 Raf-1信号通路的下游效应物。 我们已经确定 第一个Raf-1底物，丝裂原活化蛋白激酶激酶（MAP-1）， KK）。 我们已经概括了拉夫诱导的激酶级联在体外使用 纯化的组分（即，从杆状病毒过表达的活化Raf-1 SF 9细胞中的载体磷酸化并激活纯化的MAP-KK， 然后磷酸化并激活p44-MAP激酶）。 激活MAP 激酶在体外有效磷酸化c-Jun， DNA结合结构域和磷酸化的c-Myc在 氨基末端100个氨基酸。 另外，我们还发现了一起 Raf-1的表达与MAP激酶的激活无关。 在 Jurkat T细胞Raf诱导白细胞介素-2产生，而不伴随 细胞外信号调节激酶激活。 最后，我们展示了 紫外线（UV）和Raf-1诱导磷酸化（在 丝氨酸63和73）和体内c-Jun的活化。 显性阴性 Raf突变体阻断Ras和Src诱导的c-Jun磷酸化。 位点，表明在该信号通路中需要Raf-1。 的 显性负性Raf突变体也阻断Src-、Ras-、UV-、佛波酯- 以及由Ap-1/Ets或SRE驱动的报告基因的血清诱导表达 结合位点。 此外，显性负性MAP激酶和Jun突变体 阻断激活的Raf-induced转化NIH 3 T3细胞。 所以我们 结果表明，Raf-1在线性信号转导中起作用 以Src-Ras-Raf-1-MAP-KK-MAP激酶-c-Jun/Ets.

During the past year we have made considerable progress in identifying downstream effectors of the Raf-1 signalling pathway. We have identified the first Raf-1 substrate, mitogen-activated protein-kinase-kinase (MAP- KK). We have recapitulated the Raf-induced kinase cascade in vitro using purified components (i.e., activated Raf-1 overexpressed from baculovirus vectors in SF9 cells phosphorylated and activated purified MAP-KK which then phosphorylated and activated p44-MAP kinase). This activated MAP kinase efficiently phosphorylated c-Jun in vitro at residues adjacent to the DNA binding domain and phosphorylated c-Myc at residues within the amino terminal 100 amino acids. In addition, we have uncovered an action of Raf-1 that appears to be independent of MAP kinase activation. In Jurkat T-cells Raf induced interleukin-2 production without concomitant extracellular signal-regulated kinase activation. Lastly, we have shown that ultraviolet light (UV) and Raf-1 induce the phosphorylation (at serines 63 and 73) and activation of c-Jun in vivo. A dominant-negative Raf mutant blocked Ras- and Src-induced phosphorylation of c-Jun at these sites, indicating a requirement for Raf-1 in this signalling pathway. The dominant-negative Raf mutant also blocked Src-, Ras-, UV-, phorbol ester- and serum-induced expression of a reporter gene driven by Ap-1/Ets or SRE binding sites. Furthermore, dominant-negative MAP kinase and Jun mutants blocked activated Raf-induced transformation of NIH3T3 cells. Thus, our results indicate that Raf-1 functions in a linear signal transduction pathway with the order Src-Ras-Raf-1-MAP-KK-MAP kinase-c-Jun/Ets.