Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor
Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor
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DOI：
10.1074/jbc.m006087200
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发表时间：
2000-10-13
影响因子：
4.8
通讯作者：
Hancock, JF
中科院分区：
文献类型：
--
作者：
Clyde-Smith, J;Silins, G;Hancock, JF
Ras蛋白在酪氨酸激酶和G蛋白偶联受体下游的信号转导途径中作为分子开关起作用。Ras通过鸟嘌呤核苷酸交换因子（GEFs）从非活性GDP结合状态转换为活性GTP结合状态。我们在这里报道了RasGRP 2的克隆和表征，RasGRP 2是最近克隆的RapGEF（CalDAG-GEFI）的一种较长的选择性剪接形式。RasGRP 2的一个独特特征是它通过N-末端肉豆蔻酰化和棕榈酰化的组合靶向质膜。在体内，RasGRP 2选择性地催化N-和Ki-Ras上的核苷酸交换，但不催化Ha-Pas上的核苷酸交换。RasGRP 2也催化Rap 1上的核苷酸交换，但这种RapGEF活性不如与CalDAG-GEFI相关的活性强。RasGRP 2对N-Ras的核苷酸交换活性被二酰基甘油刺激，并被钙抑制。甘油二酯和钙的作用是相加的，但并不伴随着任何可检测到的变化，在亚细胞定位的RasGRP 2。相比之下，CalDAG-GEFI主要定位于胞质溶胶，并且在体内缺乏Pas交换活性。然而，长时间暴露于佛波醇酯或在血清中生长，导致CalDAG-GEFI定位于细胞膜并恢复Pas交换活性。在转染野生型N-Ras的NIH 3 T3细胞中表达RasGRP 2或CalDAG-GEFI导致生长速率加快，但不导致形态转化。因此，在适当的生长条件下，CalDAG-GEFI和RasGRP 2是双重特异性Ras和Rap交换因子。
Ras proteins operate as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDP-bound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs), We report here the cloning and characterization of RasGRP2, a longer alternatively spliced form of the recently cloned RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of N-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and Ki-Ras, but not Ha-Pas. RasGRP2 also catalyzes nucleotide exchange on Rap1, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 toward N-Ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but are not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Pas exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Pas exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH3T3 cells transfected with wild type N-Ras results in an accelerated growth rate but not morphologic transformation. Thus, under appropriate growth conditions, CalDAG-GEFI and RasGRP2 are dual specificity Ras and Rap exchange factors.