Identification of a novel domain of Ras and Rap1 that directs their differential subcellular localizations
Identification of a novel domain of Ras and Rap1 that directs their differential subcellular localizations
复制标题
DOI：
10.1074/jbc.m314169200
复制
发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Kataoka, T
中科院分区：
文献类型：
--
作者：
Nomura, K;Kanemura, H;Kataoka, T
小的GTd-Ha-Ras和Rap 1A表现出高度的相互序列同源性，并共享各种靶蛋白。然而，它们发挥不同的生物学功能，并表现出差异的亚细胞定位; Rap 1A主要定位在核周区域，包括高尔基体和内体，而Ha-Ras主要定位在质膜。在这里，我们已经确定了Rap 1A的一个小区域，这是至关重要的核周定位。对Ha-Ras-Rap 1A嵌合体的分析表明Ha-Ras在46-101位氨基酸上被Rap 1所取代，并表现出核周定位。随后的突变研究表明，Ha-Ras的85-89位的5个氨基酸内的Rap 1A型取代，如NNTKS 85 - 89 TAQST、NN 85 - 86 TA和TKS 87 - 89 QST，足以诱导Ha-Ras的核周定位。相反，该区域周围残基的取代，如FAI 82 - 84 YSI和FEDI 90 - 93 FNDL，对Ha-Ras的质膜定位没有影响。由Rap 1A的氨基酸1-134和Ha-Ras的134-189组成的嵌合构建体，具有Ha-Ras的棕榈酰化和法尼基化位点，表现出与Rap 1A一样的核周定位。将Ha-Ras型取代引入该嵌合构建体的氨基酸85-89（TAQST 85 - 89 NNTKS）中导致其主要亚细胞定位位点从核周区改变至质膜。这些结果表明，以前未知的结构域跨越氨基酸85-89的Rap 1A发挥了关键作用，在其核周定位。此外，该结构域主要作用于Ha-Ras的COOH末端脂质修饰，这被认为是质膜定位所必需的和足够的。
The small GTPase Ha-Ras and Rap1A exhibit high mutual sequence homology and share various target proteins. However, they exert distinct biological functions and exhibit differential subcellular localizations; Rap1A is predominantly localized in the perinuclear region including the Golgi apparatus and endosomes, whereas Ha-Ras is predominantly localized in the plasma membrane. Here, we have identified a small region in Rap1A that is crucial for its perinuclear localization. Analysis of a series of Ha-Ras-Rap1A chimeras shows that Ha-Ras carrying a replacement of amino acids 46-101 with that of Rap1 exhibits the perinuclear localization. Subsequent mutational studies indicate that Rap1A-type substitutions within five amino acids at positions 85-89 of Ha-Ras, such as NNTKS85-89TAQST, NN85-86TA, and TKS87-89QST, are sufficient to induce the perinuclear localization of Ha-Ras. In contrast, substitutions of residues surrounding this region, such as FAI82-84YSI and FEDI90-93FNDL, have no effect on the plasma membrane localization of Ha-Ras. A chimeric construct consisting of amino acids 1-134 of Rap1A and 134-189 of Ha-Ras, which harbors both the palmitoylation and farnesylation sites of Ha-Ras, exhibits the perinuclear localization like Rap1A. Introduction of a Ha-Ras-type substitution into amino acids 85-89 (TAQST85-89NNTKS) of this chimeric construct causes alteration of its predominant subcellular localization site from the perinuclear region to the plasma membrane. These results indicate that a previously uncharacterized domain spanning amino acids 85-89 of Rap1A plays a pivotal role in its perinuclear localization. Moreover, this domain acts dominantly over COOH-terminal lipid modification of Ha-Ras, which has been considered to be essential and sufficient for the plasma membrane localization.