Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-Ras
Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-Ras
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DOI：
10.1074/jbc.m206956200
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发表时间：
2003-05-23
影响因子：
4.8
通讯作者：
Buss, JE
中科院分区：
文献类型：
--
作者：
Baker, TL;Zheng, H;Buss, JE
H-Ras显示GTP结合和棕榈酸周转的动态循环。GTP结合明显地与活化偶联，但棕榈酰化的COOH末端是否参与信号传导，特别是当受到膜束缚时，尚不清楚。作为比较膜结合的脂质修饰的H-Ras的COOH末端的方式，测量NIH 3 T3细胞中各种形式的H-Ras的棕榈酸酯去除率。在细胞（H-RasWT）或显性阴性（H-Ras 17 N）形式中，去棕榈酰化发生缓慢（t（12）类似于2.4 h），而在致癌H-Ras 61 L或H-RasR 12，T59中发生更快（t（12）类似于1 h）。结合这一数据与GTP结合的测量，棕榈酸半衰期的H-Ras在完全GTP结合状态估计为小于10分钟。缓慢的棕榈酸去除细胞H-Ras没有解释封存在小窝，无论是细胞或致癌H-Ras显示对齐小窝免疫荧光。相反，虽然它有更快的棕榈酸酯去除，致癌H-Ras位于相同的分数作为H-RasWT在四种类型的密度梯度，并保持完全膜结合。因此，尽管致癌和细胞H-Ras似乎在相似的位置，但脱酰化的速率不同。相反，这些结果表明，酰基蛋白硫酯酶更容易接近致癌H-Ras，因为其COOH末端对膜的构象被改变。这种以前未检测到的差异可能有助于产生独特的效应器相互作用和致癌H-Ras的信号传导。
H-Ras displays dynamic cycles of GTP binding and palmitate turnover. GTP binding is clearly coupled to activation, but whether the palmitoylated COOH terminus participates in signaling, especially when constrained by membrane tethering, is unknown. As a way to compare COOH termini of membrane-bound, lipid-modified H-Ras, palmitate removal rates were measured for various forms of H-Ras in NIH 3T3 cells. Depalmitoylation occurred slowly (t(1/2) similar to2.4 h) in cellular (H-RasWT) or dominant negative (H-Ras17N) forms and more rapidly (t(1/2) similar to1 h) in oncogenic H-Ras61L or H-RasR12, T59. Combining this data with GTP binding measurements, the palmitate half-life of H-Ras in the fully GTP-bound state was estimated to be less than 10 min. Slow palmitate removal from cellular H-Ras was not explained by sequestration in caveolae, as neither cellular nor oncogenic H-Ras showed alignment with caveolin by immunofluorescence. Conversely, although it had faster palmitate removal, oncogenic H-Ras was located in the same fractions as H-RasWT on four types of density gradients, and remained fully membrane-bound. Thus the different rates of deacylation occurred even though oncogenic and cellular H-Ras appeared to be in similar locations. Instead, these results suggest that acylprotein thioesterases access oncogenic H-Ras more easily because the conformation of its COOH terminus against the membrane is altered. This previously undetected difference could help produce distinctive effector interactions and signaling of oncogenic H-Ras.