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 （MAPKK）. 我们已经概括了Raf-induced激酶级联在体外 使用纯化的组分（即，活化的Raf-1过表达于 SF 9细胞中的杆状病毒载体磷酸化并活化纯化 然后磷酸化并激活p44-MAP激酶）。 我们有 现在扩展了这些实验，并确定了Raf-specific MAPKK的磷酸化位点。 为了了解激活机制 MAPKK 1的Ser 217和Ser 221分别是MAPKK的两个位点， p74 raf-1磷酸化。 这代表了第一个特征 被这种原癌基因产物磷酸化的位点。 Ser 217和 Ser 221位于催化结构域的一个区域，在该区域中活化的 定位了几种其它蛋白激酶的磷酸化位点。 在MAPKK家族成员中，该区域是最保守的，这表明 该家族的所有成员都是通过磷酸化 这些网站。 一个“激酶死亡”的MAPKK 1突变体被磷酸化， 与野生型酶相同的残基，确定两个位点都是 通过p74 raf-1直接磷酸化，而不是通过自身磷酸化。 只 MAPKK 1的二磷酸化形式（在Ser 217和Ser 218处磷酸化）被抑制。 Ser 221），即使当磷酸化的化学计量被检测到， p74 raf-1表达较低，表明这些位点之一的磷酸化 是限速的，第二种蛋白的磷酸化 迅速 Ser 217和Ser 221在体内均被磷酸化， 用神经生长因子刺激PC 12细胞10 min。 分析其中Ser 217或Ser 221改变的MAPKK 1突变体 谷氨酸，并发现，最大限度地灭活 活化的MAPKK 1需要两种丝氨酸的去磷酸化，显示 任何一个残基磷酸化都足以最大程度地 activation.

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 (MAPKK). 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 MAPKK which then phosphorylated and activated p44-MAP kinase). We have now extended these experiments and determined the Raf-specific phosphorylation site on MAPKK. To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A "kinase-dead" MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1 and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate-limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.