Willumsen B M, Cox A D, Solski P A, Der C J, Buss J E
Department of Molecular Cell Biology, University of Copenhagen, Denmark.
Oncogene. 1996 Nov 7;13(9):1901-9.
Although it is well-established that modification of Ras by farnesol is a critical step for its membrane association and transforming activity, the contribution of other C-terminal sequences and palmitate modification to Ras localization and function remains unclear. We have characterized H-Ras mutant proteins with alterations in the palmitoylated cysteines or in sequences flanking these residues. We found that non-palmitoylated proteins were impaired not only in membrane association but also in transforming activity. Mutations which drastically altered residues adjacent to the palmitoylated cysteine did not abolish palmitoylation. However, despite continued lipid modification the mutant proteins failed to bind to plasma membranes and instead accumulated on internal membranes and, importantly, were not transforming. Addition of an N-terminal myristoylation signal to these defective mutants, or to proteins entirely lacking the C-terminal 25 residues restored both plasma membrane association and transforming activity. Thus, H-Ras does not absolutely require prenylation or palmitoylation nor indeed its hypervariable domain in order to interact with effectors that ultimately cause transformation. However, in this native state, the C-terminus appears to provide a combination of lipids and a previously unrecognized signal for specific plasma membrane targeting that are essential for the correct localization and biological function of H-Ras.
尽管法尼基醇对Ras的修饰是其膜结合和转化活性的关键步骤已得到充分证实，但其他C末端序列和棕榈酰化修饰对Ras定位和功能的作用仍不清楚。我们对棕榈酰化半胱氨酸或这些残基侧翼序列发生改变的H-Ras突变蛋白进行了表征。我们发现，非棕榈酰化蛋白不仅在膜结合方面受损，而且在转化活性方面也受损。大幅改变与棕榈酰化半胱氨酸相邻残基的突变并未消除棕榈酰化。然而，尽管持续进行脂质修饰，突变蛋白仍无法结合到质膜上，而是在内膜上积累，重要的是，它们没有转化能力。在这些缺陷突变体或完全缺乏C末端25个残基的蛋白上添加N末端肉豆蔻酰化信号，可恢复质膜结合和转化活性。因此，H-Ras与最终导致转化的效应器相互作用时，并不绝对需要异戊二烯化或棕榈酰化，实际上也不需要其高变区。然而，在这种天然状态下，C末端似乎提供了脂质组合以及一个以前未被认识的特定质膜靶向信号，这些对于H-Ras的正确定位和生物学功能至关重要。
