Derivatives of activated H-ras lacking C-terminal lipid modifications retain transforming ability if targeted to the correct subcellular location
Derivatives of activated H-ras lacking C-terminal lipid modifications retain transforming ability if targeted to the correct subcellular location
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DOI：
10.1038/sj.onc.1200908
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发表时间：
1997-02-27
期刊：
影响因子：
8
通讯作者：
Donoghue, DJ
中科院分区：
文献类型：
--
作者：
Hart, KC;Donoghue, DJ
为了检测uas在不存在脂质修饰的情况下激活信号转导途径的能力，使用禽传染性支气管炎病毒（IBV）的El蛋白的第一个跨膜结构域（其含有顺式高尔基体靶向信号）构建了将ras（WT）或活化的ras（61 L）靶向细胞膜作为整合膜蛋白的融合蛋白。高尔基体靶向的衍生物的活化ras是完全无活性的转化试验。然而，当在病灶形成测定中检查时，用含有导致质膜定位的突变El靶向序列的vas（61 L）的衍生物观察到NIH 3 T3细胞的转化。去除CAAX基序中和上游的脂质修饰位点并没有废除质膜定位的ras（61 L）衍生物的转化活性，表明这些脂质修饰对于ras活性不是必需的，只要蛋白质正确定位于质膜。有趣的是，完整膜形式的vas（61 L）的活性严格依赖于跨膜结构域锚区和vas编码序列之间的最小距离。仅具有3个氨基酸的接头的衍生物是无活性的，而11个或22个氨基酸的接头足以恢复转化活性。这些结果表明：（1）靶向高尔基体膜的活化的ras不能引起转化;（2）C-末端的脂质修饰对于质膜锚定的ras（61 L）衍生物的转化活性不是必需的，并且主要起靶向功能;（3）跨膜结构域可以有效地取代通常将UAS靶向质膜内表面的C-末端修饰，表明ras（61 L）不需要像正常脂化所允许的那样从膜上可逆地解离;和（4）为了正确地发挥功能，存在ras蛋白必须驻留在离质膜的临界距离。
To examine the ability of uas to activate signal transduction pathways in the absence of lipid modifications, fusion proteins were constructed that target ras(WT) or activated ras(61L) to cellular membranes as integral membrane proteins, using the first transmembrane domain of the El protein of avian infectious bronchitis virus (IBV), which contains a cis-Golgi targeting signal. Golgi-targeted derivatives of activated ras were completely inactive in transformation assays. However, when examined in focus formation assays, transformation of NIH3T3 cells were seen with derivatives of vas(61L) containing a mutated El targeting sequence that results in plasma membrane localization. Removal of the lipid modification sites in and upstream of the CAAX motif did not abrogate the transforming activity of plasma membrane-localized ras(61L) derivatives, indicating that these lipid modifications are not essential for ras activity, as long as the protein is correctly localized to the plasma membrane. Interestingly, the activity of integral membrane versions of vas(61L) was strictly dependent on a minimum distance between the transmembrane domain anchor region and the coding sequence of vas. Derivatives with only a 3-amino acid linker were inactive, while linkers of either 11- or 22-amino acids were sufficient to restore transforming activity. These results demonstrate that: (1) activated ras targeted to Golgi membranes is unable to cause transformation; (2) lipid modifications at the C-terminus are not required for the transforming activity of plasma membrane-anchored ras(61L) derivatives, and serve primarily a targeting function; (3) a transmembrane domain can effectively substitute for C-terminal modifications that would normally target uas to the inner surface of the plasma membrane, indicating that ras(61L) does not need to reversibly dissociate from the membrane as might be allowed by the normal lipidation; and (4) in order to function properly, there exists a critical distance that the ras protein must reside from the plasma membrane.