摘要
研究背景及目的
白血病是最常见的儿童恶性肿瘤之一,约占儿童肿瘤的1/3。尽管白血病的发病原因目前尚不清楚,已有研究显示一些产前染色体和基因异常如21三体和TEL-AML重排有增加儿童患白血病风险的倾向,而且有关双胞胎白血病患者的研究显示白血病的发生可以起始于子宫内阶段。Ras是一种膜联小分子GTP酶,在细胞信号转导中发挥重要作用。Rras基因的12、13密码子的突变可引起Ras信号的增强,从而导致Rras蛋白的持续激活,引起细胞增殖分化、恶性转化等改变。Pten是PI3K-Akt信号通路的负性调节蛋白,在细胞周期、系别分化、迁移和造血干细胞、祖细胞的功能中发挥重要作用。目前研究表明儿童白血病中存在癌性Kras的表达和Pten的缺失等基因突变。
因而我们提出一种假设:癌性Kras基因表达和Pten基因的缺失这两种基因突变可能作为早期事件发生在胚胎从而对出生后白血病的发生发挥作用。但目前存在的癌性Kras基因表达和Pten基因的缺失的小鼠模型研究大多应用成鼠的骨髓细胞,而为检验这一假设成立与否,需建立一种于胚胎阶段始可引起造血系统该两种基因突变发生的小鼠模型。
Cre转基因小鼠可敲除特异组织中两侧带有Loxp位点的基因序列,而Vav-iCre (?)、鼠中Cre基因的表达由Vav基因控制。Vav基因主要表达于成鼠的造血系统及胚胎的造血干细胞来源的造血祖细胞,因而Vav-iCre中重组酶主要作用于造血器官的造血细胞,是用来建立胚胎阶段始造血系统特异基因表达或缺失的有效工具。Kras基因在白血病中最常见的突变之一为Kras外显子1的由密码子12编码的甘氨酸突变为天冬氨酸,LSL-KrasG12D小鼠则是在该突变的上游包含有一个两端带有loxp的转录终止密码子,可被Cre重组酶切除,从而导致突变的癌性Kras基因(KrasG12D+)的表达。Pten基因的外显子5为编码磷酸酶的结构域,被认为是Pten基因发挥作用的重要结构域。条件性Pten敲除小鼠(Ptenf/f)则是在小鼠Pten基因外显子5的两端插入loxp序列,可被Cre重组酶敲除,从而导致Pten基因的功能缺失。
本研究旨在利用LSL-KrasG12D小鼠、Ptenf/f和Vav-iCre小鼠平行建立KrasG12D+表达和Pten-/-这两种胚胎阶段始血液系统非病毒致特异基因突变的小鼠模型,获得KrasG12D+或Pten稳定一致表达或缺失的基因型小鼠,观察其不同表型,探索KrasG12D+表达或Pten-/-对胚胎造血及出生后白血病发生的作用,为深入认识儿童白血病的发生机制及可能的防治工作提供动物实验依据。本研究主要分为以下两个部分:
第一部分:Kras G12D+表达对小鼠白血病发生及胚胎造血的影响
方法
1.将LSL-KrasG12D小鼠与Vav-iCre小鼠杂交,在子鼠21天时分笼编号,取部分耳组织提取DNA,通过PCR方法及基因测序方法鉴定子鼠基因型。
2.提前一晚将LSL-KrasG12D母鼠与Vav-iCre小鼠放置于一笼拟交配,于发现母鼠有阴道栓的清晨计为胚胎第0.5日(E0.5),分别于E14.5和E15.5取小鼠胚胎,对比观察组、对照组胚胎形态差异,并对正中矢状面切片固定行HE染色,观察对比胎肝组织学变化;取胎肝细胞行流式细胞学检查,检测细胞表面抗体Ter119、CD71的表达,分析观察组、对照组胎肝细胞红系细胞分化的差异。
结果
1. LSL-KrasG12D小鼠与Vav-iCre小鼠杂交后,于48只子鼠中,PCR基因型鉴定结果示Kraswt;Vav-Cre-基因型小鼠17只,占35.24%,LSL-KrasG12D基因型小鼠16只,占33.33%,Kraswt;Vav-Cre+基因型小鼠15只,占31.25%,KrasG12D+基因型小鼠(拟观察组)0只,各基因型发生率不符合孟德尔遗传定律。5只LSL-KrasG12D基因型母鼠及15只Vav-Cre+基因型小鼠的KrasG12D基因突变测序结果示5只LSL-KrasG12D基因型母鼠均含有KrasG12D突变,而Vav-Cre+基因型小鼠不含该突变。
2.从形态学上与Kraswt;Vav-Cre+基因型小鼠的胎肝相比,E14.5的KrasG12D+基因型小鼠胚胎胎肝颜色苍白,并可伴有点状出血点,E15.5的胚胎则出现脑部或弥漫性出血。E15.5胚胎矢状面切片HE染色示KrasG12D+小鼠胎肝肝叶中央部位细胞轻度减少,远端部位有核红细胞增多,伴有严重的细胞坏死。
3.流式细胞学检测E14.5的KrasG12D+基因型小鼠胚胎的新鲜胎肝细胞表面CD71、Ter119的表达结果示Ter119-CD71+细胞群比例为(8.20±4.39)%,较.Kraswt;Vav-Cre+基因型小鼠胚胎的(3.66±0.62)%明显增高,两组相比有显著的统计学意义(P<0.001)。
小结
1.LSL-KrasG12D小鼠与Vav-iCre小鼠杂交后不能获得成活的KrasG12D+基因型小鼠。
2.KrasG12D+基因型小鼠胚胎于E14.5-E15.5因发生脑部出血或弥漫性出血而死亡。
3.KrasG12D+基因型小鼠胚胎胎肝中红系原始细胞及祖细胞比率增高。
第二部分:Pten表达缺失对小鼠白血病发生及胚胎造血的影响
方法
1.将Ptenf/f与Vav-iCre小鼠杂交,获得PtenΔ/+;Vav-Cre+基因型(Pten+/-)小鼠,再次与Ptenf/f小鼠杂交,行基因型鉴定,分析不同基因型表达是否符合孟德尔遗传定律。将小鼠分为Pten-/-(PtenΔ/Δ;Vav-Cre+)基因型(观察组)、Pten+/-(PtenΔ/+;Vav-Cre+)基因型(对照组)和Ptenf/f/Ptenf/+基因型(对照组)三组。每日观察小鼠状态,于50天取小鼠外周血行全血细胞计数,观察细胞分类、比例变化。于观察组小鼠病重时对小鼠行安乐死,取外周血细胞行细胞离心涂片、Giemsa染色,取脾脏、肝脏、胸腺组织固定、HE染色,观察细胞形态,分析肿瘤类型,对骨髓细胞或胸腺细胞行流式细胞学检查,检测其细胞表面抗体CD19、Thy1、CD45、CD4、CD8等表达,进一步分析肿瘤类型,同时对照组小鼠做相同处理,分析观察组小鼠和对照组小鼠生存曲线的差异。
2.小鼠骨髓细胞移植实验:取4×106Pten-/-基因型小鼠的胸腺细胞或骨髓细胞(CD45.2),通过小鼠尾静脉注射入6-8周龄的亚致死量照射(650cGy)的移植受体C57BL/6J (CD45.1/CD45.2)小鼠,每日观察小鼠状态,于小鼠病重时对小鼠行安乐死,提取新鲜的骨髓或脾脏细胞,通过流式细胞学检测CD45.1、 CD45.2及CD3、CD4、CD8表达,CD45.1-CD45.2+细胞群为植入细胞,含有2.5%以上的CD45.1-CD45.2+细胞认为是有效移植。
3.提前一晚将Pten+/-母鼠与Vav-iCre小鼠放置于一笼拟交配,于发现母鼠有阴道栓的清晨计为胚胎第0.5日(E0.5),于E14.5取小鼠胚胎,行基因型鉴定,观察胚胎形态有无差异,并对胎肝细胞行流式细胞学检查,检测细胞表面抗体,分析观察组、对照组胎肝造血祖细胞组成成分比例的差异。
结果
1. Ptenf/f小鼠与Vav-iCre小鼠杂交后,获得50%Pten+/基因型小鼠,将6只Pten+/-小鼠分别与Vav-iCre小鼠杂交后,于61只子鼠中,PCR基因型鉴定结果示Pten-/-基因型小鼠13只(观察组),占21.31%,Pten+/-基因型小鼠17只,占27.87%, Ptenf/f基因型小鼠15只,占25.00%,Ptenf/+基因型小鼠16只,占26.23%,各基因型发生率符合孟德尔遗传定律。Pten-/-基因型小鼠中位生存时间为82天,而Pten+/-基因型小鼠的中位生存时间大于200天,两组相比其差异具有显著性统计学意义(P<0.001)。
2.于50天取小鼠外周血行全血细胞计数,结果显示Pten-/-基因型小鼠白细胞数量较Pten+/基因型小鼠明显增高,具有显著统计学意义(P<0.05)。
3.从形态学上与Pten+/-基因型小鼠相比,Pten-/-基因型小鼠出现弓背、呼吸抑制及体重减轻,解剖学检查结果示胸腺、脾脏和肝脏体积增大、重量增加(P<0.05)。
4.与Pten+/-基因型小鼠相比,Pten-/-基因型小鼠胸腺HE染色示胸腺出现淋巴母细胞浸润,正常组织结构消失;脾脏、肝脏切片HE染色示脾脏、肝脏出现白血病细胞浸润,正常组织结构破坏;外周血细胞离心涂片Giemsa染色示外周血中出现原始淋巴细胞浸润。
5.应用流式细胞学检测胸腺细胞表面抗体CD19(B细胞)和Thy1(T细胞)的表达,结果示其主要为CD19-Thy+((80.2±2.7)%),而后检测CD45+的胸腺细胞群表面抗体CD4和CD8表达,结果示3只小鼠胸腺肿瘤细胞为CD4+CD8+(1/2),1只为CD8+CD4-(1/6),另外两只为CD4+CD8-(1/3).
6.小鼠的骨髓细胞移植实验结果示4×106的Pten-/-基因型小鼠胸腺细胞或骨髓细胞经静脉注射入移植受体C57BL/6J(CD45.1/CD45.2)小鼠后,于2-16周受体小鼠发病,表现基本同Pten-/-小鼠,流式细胞学检测骨髓细胞中CD45.1-CD45.2+细胞群均>5%((24.4+3.1)%),且该群细胞群表面抗体CD3+。
7.应用流式细胞学检测小鼠胚胎胎肝细胞表面的lin、Scal-1、c-kit、CD34和FcRⅡ/Ⅲ等抗体表达,结果示与Pten++/-基因型小鼠相比,Pten-/-基因型小鼠胚胎胎肝细胞中富含造血干细胞的LSK (lin-Scal-l+c-kit+)细胞群无明显变化,而造血祖细胞(HPC)中的共同髓系祖细胞群(CMP)增高,共同淋系祖细胞群(CLP)减少,两组差异具有显著性统计学意义(P<0.05,P<0.01)。
小结
1. Ptenf/f小鼠与Vav-iCre小鼠杂交后,成功获得长期正常生存的Pten+/-基因型小鼠。Pten+/-基因型小鼠与Ptenf/f小鼠杂交后,成功获得可存活的Pten-/-基因型小鼠。Pten-/-基因型小鼠中位生存时间较Pten+/-基因型小鼠的中位生存时间明显缩短。
2. Pten-/基因型小鼠发生肝脾肿大、胸腺增大,细胞形态学及流式细胞学显示急性T淋巴细胞白血病/淋巴瘤(T-ALL/L)特性。
3. Pten-/-基因型小鼠发生的T-ALL/L具有可移植性。
4. Pten-/-基因型小鼠的胚胎胎肝造血祖细胞中CMP增高,而CLP减少。
结论
1.利用Vav-iCre转基因小鼠诱导KrasG12D+在发育阶段表达的KrasG12D+基因型小鼠于E14.5-E15.5死亡,提示该小鼠模型不能用于解释说明胚胎期KrasG12D+表达可导致出生后白血病发生这一问题。
2.胚胎期KrasG12D表达阻碍小鼠胎肝红系祖细胞向成熟红细胞的分化,导致KrasG12D+小鼠胚胎出现贫血。
3.胚胎期始Pten基因的单倍缺失不影响小鼠的表型,而Pten-/-小鼠发生可移植的T-ALL/L。
4.胚胎期Pten表达缺失除了在祖细胞水平引起淋系和髓系的交错变化外,对总的胚胎造血无明显影响。
Background and Objective
Leukemia is the most common children malignancy, accounting for almost30%of children cancer. Although the causes of leukemia are largely unknown, a few prenatal chromosomal and genetic alterations, such as trisomy21(Down syndrome) and TEL-AML1rearrangements have been associated with an increased risk of children leukemia, and studies of twin leukemia have revealed that leukemia can be initiated in utero. Ras are membrane-associated small GTPases and play a critical role in transducing signals, and the mutations at codons12and13cause hyperactive Ras signaling, which leads to constitutive activation of the Ras protein and affect the cell proliferation/differentiation, survival and malignant transformation. Pten (phosphatase and tensin homologue), as a negative regulator of the PI3K-Akt pathway, is involved in cell cycling, lineage commitment, mobilization, and hematopoietic stem/progenitor cell function. Both gain-of-functional Kras mutation(s) and loss of Pten expression have been implicated in children leukemia.
Taken together, we raised the hypothesis that both oncogenic K-ras mutation(s) and Pten deficiency could occur in utero as an early event which contributes to postnatal leukemogenesis. Most existing murine models on these two genetic alterations use adult bone marrow cells and are not appropriate to address this hypothesis.
Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific tissue. In Vav-iCre transgenic mouse, the iCre transgene is expressed under the control of murine Vav gene regulatory elements. The expression of Vav gene is exclusively restricted to hematopoietic cells in adult mice and may be in hematopoietic stem cell (HSC)-derived progenitors in earliest prenatal. Therefore, Vav-iCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs and the Vav-iCre transgene mice are effective tools for constructing fetal-stage-blood-lineage specific nonviral-based genetic modified murine models. In LSL-KrasG12D mice, there is a transcriptional termination codon flanked by loxP sites upstream of a mutation of glycine to aspartic acid encoded by codon12in exonl. Excision of the stop cassette by iCre recombinase allows the expression of oncogenic Kras. The conditional Pten konckout mice (Ptenf/f) were inserted two loxP sequences on either side of the exon5, which encode the phosphatase domain and erased by iCre recombinase leading to disruption of Pten.
In this study, by using LSL-KrasG12D mice, Ptenf/f mice, and Vav-iCre transgene mice, we sought to constructed two fetal-stage-blood-lineage specific nonviral-based genetic modified murine models, and obtain stable and uniformed overexpression or deletion of the gene of interest, to evaluate the effect(s) of deregulated KrasG12D and Pten during fetal hematopoiesis and their potential roles in postnatal leukemogenesis. This study can be divided into two parts:
Part Ⅰ:Effects of oncogenic Kras G12D+in murine postnatal leukemogenesis and fetal hematopoiesis
Methods
1. Crossed LSL-KrasG12D mice and Vav-iCre mice, extract DNA from ear tissues of mice at21days after birth, identified the genetypes of offsprings by PCR and gene sequencing.
2. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5and E15.5, carefully dissect and gather the embryos, HE stain the parasagittal section, observe the differences on gross and histological morphology; collect fresh fetal liver cells and check the expressions of CD71, Ter119by flow cytometric analysis, compare the percentage of primitive progenitor cells and proerythroblasts.
Results
1. Among the first48offsprings, there are17Kraswt;Vav-Cre-mice (35.24%),16LSL-KrasG12D mice (33.33%) and15Kraswt;Vav-Cre+mice (31.25%), but no viable KrasG12D+;Vav-iCre+pups were identified, which is not consistant with the Mendelian inheritance. No KrasG12D mutation was found in15Vav-iCre+littermate mice by sequencing on purified genotyping PCR products.
2. Compared with Kraswt;Vav-Cre+embryos, the E14.5KrasG12D+embryos have pale-colored liver and small hemorrhage foci, the E15.5KrasG12D+embryos appear brain or extensive systemic hemorrhage. In hematoxylin and eosin (HE) stained fetal liver parasagittal section from E15.5KrasG12D+embryos, mild decrease in cellularity in the proximal portion of and increased erythroblasts in the distal portion of the KrasG12D+hepatic lobe were observed. Moreover, severe cell death was observed in the distal portion of the KrasG12D+hepatic lobe.
3. Cells isolated from E14.5Kraswt;Vav-Cre+or KrasG12D+fetal livers were stained with anti-CD71and anti-Ter119for flow cytometric analysis (FCAS). It displayed an increased Ter119-CD71+populations ((8.20±4.39)%) in KrasG12D+fetal livers compared with (3.66±0.62)%in Kraswt;Vav-Cre+fetal livers. The difference is significant (P<0.001)。
Summary
1. No live KrasG12D+; Vav-iCre+pups were detected by crossing LSL-KrasG12Dmice and Vav-iCre mice.
2. KrasG12D+embryos died of brain or extensive systemic hemorrhage between E14.5to E15.5.
3. KrasG12D+embryos fetal livers displayed an increased percentage in primitive progenitor cells and proerythroblasts.
Part Ⅱ:Effects of Pten deletion in murine postnatal leukemogenesis and fetal hematopoiesis
Methods
1. Crossed Ptenf/f mice with Vav-iCre to get Pten+/-mice, then crossed Pten+/-mice with Ptenf/f mice. Identified the genotypes of offsprings, and divided into observation group and control groups. Monitor mice everyday, detecte the complete blood count CBC using peripheral blood (PB) on50day after birth. Euthanize badly sick mice with carbon dioxide, and collect PB to do cytospin and Giemsa stain, fix spleen, liver, thymus tissues and HE stain, isolate bone marrow (BM) cells for FCAS. Meanwhile, dealed with the control groups mice as the same way, and analyse their median survival difference.
2. mice BM cells/thymocytes transplantation:4×106BM cells or thymocytes harvested from Pten-/-mice (CD45.2) were injected intravenously via the tail vein into sub-lethally irradiated (650cGy)6to8-week-old CD45.1/CD45.2mice. Mice were monitored everyday, and were euthanized when badly sick. Fresh cells harvested from BM and spleen were subsequently subjected to FCAS utilizing CD45.1, CD45.2and CD4/CD8antibody. The percentage of CD45.1-CD45.2+was calculated as engraftment cells, and a threshold of2.5%CD45.1-CD45.2+cells was established as a reliable preditor of positive engraftment.
3. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5, carefully dissect and gather the embryos, observe the differences in gross morphology; collect fresh fetal liver cells for FACS, compare the percentage difference of hematopoietic progenitor cells (HPC).
Results
1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice occupied50%in offsprings and live normally more than200days. Among the61offsprings by crossing Pten+/-mice with Ptenf/f mice, there are13Pten-/-mice (21.31%),17 Pten+/-mice (27.87%),15Ptenf/f mice (25.00%) and16Ptenf/+mice (26.23%), which is consistant with the Mendelian inheritance. Almost all Pten-/-mice died with a median survival of82days, which was significantly shorter than that of Pten+/-mice(P<0.001).
2. CBC showed the white blood cells (WBC,(36.22±12.46)×109) in Pten-/-mice is signicantly higher than that in Pten+/-mice (7.17±2.01)×109, P<0.05).
3. Compared with Pten-/-mice, the Pten-/-mice showed hunched posture, respiratory distress, ruffled fur and weight loss (incidence100%). Necrospy displayed the presence of a thymic mass accompanied by hepatosplenomegaly (incidence85.7%).
4. Histological examination of the thymus revealed that the normal thymic architecture was effaced and infiltrated by monomorphic lymphoblastic cells with prominent nucleoli and scant cytoplasm. Leukemic cells infiltrated organs such as spleen and liver. Variable leukemic blasts were present in the peripheral blood of diseased mice.
5. Tumor thymocytes were examined for their expression of Thyl and CD19, markers for T cells and B cells respectively. Most of these cells were Thyl+but CD19-((80.2±2.7)%). The CD45+populations were analysed using CD4and CD8, and it showed three (1/2) of the six examined tumors consisted of predominantly CD4+CD8+cells, one (1/6) contained primarily CD4-CD8+cells and the other two (1/3) consisted of CD4+CD8-cells.
6. Mice BM cells/thymocytes transplantation test showed the recipient mice were sick in2-16week after injection. FACS results showed the percentage of CD45.1-CD45.2+populations in BM of recipient mice are more than5%((24.4±3.1)%), and the populations are CD3+.
7. FACS on fetal liver cells revealed that there was no reduction in the hematopoietic stem cell (HSC) enriched lin-sca-1+c-kit+(LSK) population. However, there was an increased percentage of common myeloid progenitor (CMP) population as well as a decreased percentage of common lymphoid progenitor (CLP).
Summary
1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice were born and live normally. By crossing Pten+/-mice with Ptenf/f mice, alive Pten-/-mice were born. The median survival of Pten-/-mice was significantly shorter than that of Pten+/-mice.
2. Pten-/-mice developed into T-ALL/L ultimately.
3. The T-ALL/L occurred in Pten-/-mice was transplantable。
4. Pten-/-mice showed an increased percentage of CMP population as well as a decreased percentage of CLP in fetal liver hematopoietic progenitor cells.
Conclution
1. K-rasG12D mutation introduced by Vav-iCre during development leads to fetal lethality, suggesting that this murine model cannot be used to assess the leukemogenic ability of K-ras in utero.
2. Oncogenic KrasG12D+expression during development may block the differention from erythroid progenitor cells to mature erythrocytes, which might be the cause of anemia.
3. None of Pten+/-mice with one copy Pten deletion during fetal stage exhibited T-ALL/L, while Pten-null mice lead to transplantable T-ALL/L.
4. Pten deletion during fetal stage hardly affect the overall fetal hematopoiesis except a skewed lymphoid/myeloid development at the progenitor level。
引文
[1]Pui CH. Childhood leukemias. N Engl J Med.1995; 332:1618-30.
[2]Smith M, Ries L, Gurney J, et al. Leukemia. Cancer incidence and survival among children and adolescents:United States SEER Program 1975-1995. Pub. No.99-4649 ed. Bethesda, MD:National Cancer Institute, SEER Program; 1999. p 17-34.
[3]Orazi A, Germing U. The myelodysplastic/myeloproliferative neoplasms: myeloproliferative diseases with dysplastic features. Leukemia 2008; 22 (7):1308-19.
[4]Emanuel PD. Myelodysplasia and myeloproliferative disorders in childhood:an update. Br J Haematol 1999; 105 (4):852-63.
[5]Hasle H, Niemeyer CM, Chessells JM, et al. A pediatric approach to the WHO classification of myelodysplastic and myeloproliferative diseases. Leukemia,2003; 17 (2): 277-82.
[6]Arico M, Biondi A, Pui CH. Juvenile myelomonocytic leukemia. Blood 1997; 90 (2): 479-88.
[7]Chen B. A., Huang Z. H., ZhangX. P., et al. An epidemiological investigation of leukemia incidence between 2003 and 2007 in Nanjing, China. J Hematol Oncol 3,21
[8]Belson M, Kingsley B., and Holmes A. Risk factors for acute leukemia in children:a review. Environ Health Perspect.2007; 115:138-145.
[9]Chokkalingam AP, Buffler PA. Genetic susceptibility to childhood leukaemia. Radiat Prot Dosimetry.2008; 132:119-29.
[10]Enciso-Mora V, Hosking FJ, Sheridan E, et al. Common genetic variation contributes significantly to the risk of childhood B-cell precursor acute lymphoblastic leukemia. Leukemia.2012; 26:2212-5.
[11]Wiemels J. Perspectives on the causes of childhood leukemia. Chem Biol Interact.2012; 196:59-67.
[12]Greaves MF, Wiemels J. Origins of chromosome translocations in childhood leukaemia. Nat Rev Cancer.2003; 3:639-49,.
[13]Greaves MF, Maia AT, Wiemels JL, et al. Leukemia in twins:lessons in natural history. Blood 2003; 102:2321-2333.
[14]Bourne HR, Sanders DA, McCormick F. The GTPase superfamily:conserved structure and molecular mechanism. Nature.1991; 349:117-127.
[15]Bourne HR, Sanders DA, McCormick F. The GTPase superfamily:a conserved switch for diverse cell functions. Nature.1990; 348:125-132.
[16]Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature.1993; 366:643-654.
[17]Quilliam LA, Rebhun JF, Castro AF. A growing family of guanine nucleotide exchange factors is responsible for activation of Ras-family GTPases. Prog Nucleic Acid Res Mol Biol.2002; 71:391-444.
[18]Campbell SL, Khosravi-Far R, Rossman KL, et al. Increasing complexity of Ras signaling. Oncogene 1998; 17:1395-413.
[19]Shannon K. The Ras signaling pathway and the molecular basis of myeloid leukemogenesis. Curr Opin Hematol.1995; 2:305-308.
[20]Bos JL. Ras oncogenes in human cancer:a review. Cancer Res.1989; 49:4682-4689.
[21]von Lintig FC, Huvar I, Law P, et al. Ras activation in normal white blood cells and childhood acute lymphoblastic leukemia. Clin Cancer Res.2000; 6:1804-1810.
[22]Vardiman JW, Pierre R, Imbert M, et al.:Juvenile myelomonocytic leukaemia. In:Jaffe ES, Harris NL, Stein H, et al., eds.:Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France:IARC Press,2001. World Health Organization Classification of Tumours,3, pp 55-7.
[23]Flotho C, Valcamonica S, Mach-Pascual S, et al. RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML). Leukemia,1999; 13 (1):32-7.
[24]Side LE, Emanuel PD, Taylor B, et al. Mutations of the NF1 gene in children with juvenile myelomonocytic leukemia without clinical evidence of neurofibromatosis, type 1. Blood, 1998; 92(1):267-72.
[25]Tartaglia M, Niemeyer CM, Fragale A, et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet.2003; 34:148-150.
[26]Van Meter ME, Diaz-Flores E, Archard JA, et al. K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells. Blood.2007; 109:3945-3952.
[27]Chan IT, Kutok JL, Williams IR, et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest.2004; 113:528-538.
[28]Braun BS, Tuveson DA, Kong N, et al. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci U S A.2004; 101:597-602.
[29]Le DT, Kong N, Zhu Y, et al. Somatic inactivation of Nfl in hematopoietic cells results in a progressive myeloproliferative disorder. Blood.2004; 103:4243-4250.
[30]Largaespada DA, Brannan CI, Jenkins NA, et al. Nfl deficiency causes Ras-mediated granulocyte/macrophage colony stimulating factor hypersensitivity and chronic myeloid leukaemia. Nat Genet.1996; 12:137-143.
[31]Koera K, Nakamura K, Nakao K, et al. K-ras is essential for the development of the mouse embryo. Oncogene.1997; 15:1151-1159.
[32]Esteban LM, Vicario-Abejon C, Fernandez-Salguero P, et al. Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development. Mol Cell Biol.2001; 21:1444-1452.
[33]Umanoff H, Edelmann W, Pellicer A, et al. The murine N-ras gene is not essential for growth and development. Proc Natl Acad Sci U S A.1995; 92:1709-1713.
[34]Guerra C, Mijimolle N, Dhawahir A, et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell.2003; 4:111-120.
[35]de Boer J, Williams A, Skavdis G, et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol.2003; 33:314-325.
[36]Georgiades P, Ogilvy S, Duval H, et al. vavCre Transgenic Mice:A Tool for Mutagenesis in Hematopoietic and Endothelial Lineages. Genesis.2002; 34:251-256.
[37]Gan T, Jude CD, Zaffuto K, Ernst P. Developmentally induced Mill loss reveals defects in postnatal haematopoiesis. Leukemia.2010; 24:1732-1741.
[38]Gutierrez A, Sanda T, Grebliunaite R, et al. High frequency of PTEN, PI3K, and AKT abnormalities in T-cell acute lymphoblastic leukemia. Blood.2009; 114:647-650.
[39]Liu YL, Castleberry RP, Emanuel PD. PTEN deficiency is a common defect in juvenile myelomonocytic leukemia. Leukemia Research.2009; 33:671-677.
[40]45. Dahia PL, Aguiar RC, Alberta J, et al. PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanisms in haematological malignancies. Hum Mol Genet.1999; 8:185-93.
[41]Leslie NR, Downes CP. PTEN function:how normal cells control it and tumor cells lose it. Biochem J.2004; 382:1-11.
[42]Chow LML, Baker SJ. PTEN function in normal and neoplastic growth. Cancer Lett.2006; 241:184-96.
[43]Zhou XP, Gimm O, Hampel H, et al. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation. Am J Pathol.2000; 157:1123-8.
[44]Cantley LC, Neel BG New insights into tumor suppression:PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci USA.1999; 96:4240-5.
[45]Cully M, You H, Levine AJ, et al. Beyond PTEN mutations:the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev.2006; 6:184-92.
[46]Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT et al. PTEN maintains haemaopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature. 2006; 441:518-522.
[47]Yilmaz OH, Valdez R, Theisen BK, et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature.2006; 441:475-482.
[48]Yu H, Li Y, Gao C, et al. Relevant mouse model for human leukemia through Cre/lox-controlled myeloid-specific deletion of PTEN. Leukemia.2010; 24:1077-1080.
[49]Tsai FY, Keller G, Kuo FC, et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature.1994; 371(6494):221-6
[50]Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell.1995; 81(5):695-704
[51]Wang Q, Stacy T, Binder M, et al. Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis._Proc Natl Acad Sci.1996; 93(8):3444-9
[52]Okuda T, van Deursen J, Hiebert SW, et al. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell. 1996; 84:321-330.
[53]Maki K, Yamagata T, Asai T, et al. Dysplastic definitive hematopoiesis in AML1/EVI1 knock-in embryos. Blood.2005; 106:2147-2155.
[54]Wang Q, Stacy T, Miller JD, et al. The CBFbeta subunit is essential for CBFalpha2 (AML1) function in vivo. Cell.1996; 87:697-708.
[55]Kang CD, Do IR, Kim KW, et al. Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells. Exp Mol Med.1999; 31:76-82.
[56]Scheele JS, Ripple D, Lubbert M. The role of ras and other low molecular weight guanine nucleotide (GTP)-binding proteins during hematopoietic cell differentiation. Cell Mol Life Sci.2000; 57:1950-1963.
[57]Chida D, Miura O, Yoshimura A, Miyajima A. Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells:functional analysis of signaling molecules by retrovirus-mediated expression. Blood.1999; 93:1567-1578.
[58]Khalaf WF, White H, Wenning MJ, et al. K-Ras is essential for normal fetal liver erythropoiesis. Blood.2005; 105(9):3538-41.
[59]Pui CH, Jeha S New therapeutic strategies for the treatment of acute lymphoblastic leukaemia. Nat Rev Drug Discov.2007; 6:149-165.
[60]Larson Gedman A, et al. The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia:A report from the Children's Oncology Group. Leukemia.2009; 23:1417-1425.
[61]Maser RS, Choudhury B, Campbell PJ, et al. Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers. Nature.2007; 447:966-971.
[62]Palomero T, Sulis ML, Cortina M, et al. Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med.2007; 13:1203-1210.
[63]Guo W, Lasky JL, Chang CJ, et al. Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation. Nature.2008; 453:529-533.
[1]Bourne HR, Sanders DA, McCormick F. The GTPase superfamily:a conserved switch for diverse cell functions. Nature 348:125-132,1990.
[2]Cherfils J, Zeghouf M. Chronicles of the GTPase switch. Nat Chem Biol 7:493-495,2011.
[3]Cox AD, Der CJ. Ras history:the saga continues. Small GTPases 1:2-27,2011.
[4]Vetter IR, Wittinghofer A. The guanine nucleotide-binding switch in three dimensions. Science 294:1299-1304,2001.
[5]Bos JL, Rehmann H, Wittinghofer A. GEFs and GAPs:critical elements in the control of small G proteins. Cell 129:865-877,2007.
[6]Biou V, Cherfils J. Structural principles for the multispecificity of small GTP-binding proteins. Biochemistry 43:6833-6840,2004.
[7]Reuther GW, Der CJ. The Ras branch of small GTPases:Ras family members don't fall far from the tree. Curr Opin Cell Biol 12:157-165,2000.
[8]Diez D, Sanchez-Jimenez F, Ranea JA. Evolutionary expansion of the Ras switch regulatory module in eukaryotes. Nucleic Acids Res 39:5526-5537,2011.
[9]Jaffe AB, Hall A. Rho GTPases:biochemistry and biology. Annu Rev Cell Dev Biol 21:247-269,2005.
[10]Pertz O. Spatio-temporal Rho GTPase signaling:where are we now? J Cell Sci 123:1841-1850,2010.
[11]Ramaen O, Joubert A, Simister P, et al. Interactions between conserved domains within homodimers in the BIG1, BIG2, and GBF1 Arf guanine nucleotide exchange factors. J Biol Chem 282:28834-28842,2007.
[12]Cote JF, Vuori K. GEF what? Dock180 and related proteins help Rac to polarize cells in new ways. Trends Cell Biol 17:383-393,2007.
[13]Rossman KL, Der CJ, Sondek J. GEF means go:turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6:167-180,2005.
[14]Mahankali M, Peng HJ, Henkels KM, et al. Phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for the GTPase Rac2. Proc Natl Acad Sci USA 108: 19617-19622,2011.
[15]Tcherkezian J, Lamarche-Vane N. Current knowledge of the large RhoGAP family of proteins. Biol Cell 99:67-86,2007.
[16]Mucha E, Fricke I, Schaefer A, et al. Rho proteins of plants-functional cycle and regulation of cytoskeletal dynamics. Eur J Cell Biol 90:934-943,2011.
[17]Dransart E, Olofsson B, Cherfils J. RhoGDIs revisited:novel roles in Rho regulation. Traffic 6:957-966,2005.
[18]Behnia R, Munro S. Organelle identity and the signposts for membrane traffic. Nature 438: 597-604,2005.
[19]Hutagalung AH, Novick PJ. Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119-149,2011.
[20]Carney DS, Davies BA, Horazdovsky BF. Vps9 domain-containing proteins:activators of Rab5 GTPases from yeast to neurons. Trends Cell Biol 16:27-35,2006.
[21]Marat AL, Dokainish H, McPherson PS. DENN domain proteins:regulators of Rab GTPases. J Biol Chem 286:13791-13800,2011.
[22]Barr F, Lambright DG. Rab GEFs and GAPs. Curr Opin Cell Biol 22:461-470,2010.
[23]Barrowman J, Bhandari D, Reinisch K, et al. TRAPP complexes in membrane traffic: convergence through a common Rab. Nat Rev Mol Cell Biol 11:759-763,2010.
[24]Nordmann M, Cabrera M, Perz A, et al. The Monl-Cczl complex is the GEF of the late endosomal Rab7 homolog Ypt7. Curr Biol 20:1654-1659,2010.
[25]Itzen A, Pylypenko O, Goody RS, et al. Nucleotide exchange via local protein unfolding: structure of Rab8 in complex with MSS4. EMBO J 25:1445-1455,2006.
[26]Frasa MA, Koessmeier KT, Ahmadian MR, et al. Illuminating the functional and structural repertoire of human TBC/RABGAPs. Nat Rev Mol Cell Biol 2012.
[27]Fukuda M. TBC proteins:GAPs for mammalian small GTPase Rab? Biosci Rep 31: 159-168,2011.
[28]Clabecq A, Henry JP, Darchen F. Biochemical characterization of Rab3-GTPaseactivating protein reveals a mechanism similar to that of Ras-GAP. J Biol Chem 275:31786-31791, 2000.
[29]Aligianis IA, Johnson CA, Gissen P, et al. Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome. Nat Genet 37:221-223,2005.
[30]Pasqualato S, Cherfils J. Crystallographic evidence for substrate-assisted GTP hydrolysis by a small GTP binding protein. Structure 13:533-540,2005.
[31]D'Souza-Schorey C, Chavrier P. ARF proteins:roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 7:347-358,2006.
[32]Casanova JE. Regulation of arf activation:the sec7 family of guanine nucleotide exchange factors. Traffic 8:1476-1485,2007.
[33]Kahn RA, Bruford E, Inoue H, et al. Consensus nomenclature for the human ArfGAP domain-containing proteins. J Cell Biol 182:1039-1044,2008.
[34]Bowzard JB, Cheng D, Peng J, et al. ELMOD2 is an Ar12 GTPase-activating protein that also acts on Arfs. J Biol Chem 282:17568-17580,2007.
[35]Yudin D, Fainzilber M. Ran on tracks-cytoplasmic roles for a nuclear regulator. J Cell Sci 122:587-593,2009.
[36]Lonhienne TG, Forwood JK, Marfori M, et al. Importin-beta is a GDP-to-GTP exchange factor of Ran:implications for the mechanism of nuclear import. J Biol Chem 284: 22549-22558,2009.
[37]Schulze H, Dose M, Korpal M, et al. RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules.J Biol Chem 283: 14109-14119,2008.
[38]Cherfils J, Chardin P. GEFs:structural basis for their activation of small GTP-binding proteins. Trends Biochem Sci 24:306-311,1999.
[39]Klebe C, Prinz H, Wittinghofer A, et al. The kinetic mechanism of Ran-nucleotide exchange catalyzed by RCC1. Biochemistry 34:12543-12552,1995.
[40]Lenzen C, Cool RH, Prinz H, et al. Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm. Biochemistry 37:7420-7430,1998.
[41]Schoebel S, Oesterlin LK, Blankenfeldt W, et al. RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleotide exchange activity.Mol Cell 36: 1060-1072,2009.
[42]Boriack-Sjodin PA, Margarit SM, Bar-Sagi D, et al. The structural basis of the activation of Ras by Sos. Nature 394:337-343,1998.
[43]Mossessova E, Gulbis JM, Goldberg J. Structure of the guanine nucleotide exchange factor Sec7 domain of human arno and analysis of the interaction with ARF GTPase.Cell 92: 415-423,1998.
[44]Biou V, Aizel K, Roblin P, et al. SAXS and X-ray crystallography suggest an unfolding model for the GDP/GTP conformational switch of the small GTPase Arf6. J Mol Biol 402: 696-707,2010.
[45]Snyder JT, Worthylake DK, Rossman KL, et al. Structural basis for the selective activation of Rho GTPases by Dbl exchange factors. Nature Struct Biol 9:468-475,2002.
[46]Renault L, Guibert B, Cherfils J. Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor. Nature 426:525-530,2003.
[47]Thomas C, Fricke I, Scrima A, et al. Structural evidence for a common intermediate in small G protein-GEF reactions. Mol Cell 25:141-149,2007.
[48]Yang J, Zhang Z, Roe SM, et al. Activation of Rho GTPases by DOCK exchange factors is mediated by a nucleotide sensor. Science 325:1398-1402,2009.
[49]Uejima T, Ihara K, Goh T, et al. GDPbound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5-Vps9 system. J Biol Chem 285:36689-36697, 2010.
[50]Mossessova E, Corpina RA, Goldberg J. Crystal structure of ARF1*Sec7 complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism.Mol Cell 12:1403-1411,2003.
[51]Thomas C, Fricke I, Weyand M, et al.3D structure of a binary ROP-PRONE complex:the final intermediate for a complete set of molecular snapshots of the RopGEF reaction. Biol Chem 390:427-435,2009.
[52]Crechet JB, Poullet P, Mistou MY, et al. Enhancement of the GDP-GTP exchange of RAS proteins by the carboxyl-terminal domain of SCD25. Science 248:866-868,1990.
[53]Chardin P, Camonis JH, Gale NW, et al. Human Sosl:a guanine nucleotide exchange factor for Ras that binds to GRB2. Science 260:1338-1343,1993.
[54]Rehmann H, Arias-Palomo E, Hadders MA, et al. Structure of Epac2 in complex with a cyclic AMP analogue and RAP1B. Nature 455:124-127,2008.
[55]Freedman TS, Sondermann H, Friedland GD, et al. A Ras-induced conformational switch in the Ras activator Son of sevenless. Proc Natl Acad Sci USA 103:16692-16697,2006.
[56]Jacquet E, Vanoni M, Ferrari C, et al. A mouse CDC25-like product enhances the formation of the active GTP complex of human ras p21 and Saccharomyces cerevisiae RAS2 proteins. J Biol Chem 267:24181-24183,1992.
[57]Peng W, Xu J, Guan X, et al. Structural study of the Cdc25 domain from Ral-specific guanine-nucleotide exchange factor Ra1GPS1a. Protein Cell 2:308-319,2011.
[58]Soisson SM, Nimnual AS, Uy M, et al. Crystal structure of the Dbl and pleckstrin homology domains from the human Son of sevenless protein. Cell 95:259-268,1,998.
[59]Worthylake DK, Rossman KL, Sondek J. Crystal structure of Racl in complex with the guanine nucleotide exchange region of Tiaml. Nature 408:682-688,2000.
[60]Bouquier N, Vignal E, Charrasse S, et al. A cell active chemical GEF inhibitor selectively targets the Trio/RhoG/Racl signaling pathway. Chem Biol 16:657-666,2009.
[61]Kulkarni K, Yang J, Zhang Z, et al. Multiple factors confer specific Cdc42 and Rac protein activation by dedicator of cytokinesis (DOCK) nucleotide exchange factors. J Biol Chem 286:25341-25351,2011.
[62]Meller N, Irani-Tehrani M, Ratnikov BI, et al. The novel Cdc42 guanine nucleotide exchange factor, ziziminl, dimerizes via the Cdc42-binding CZH2 domain. J Biol Chem279:37470-37476,2004.
[63]Gu Y, Li S, Lord EM, et al. Members of a novel class of Arabidopsis Rho guanine nucleotide exchange factors control Rho GTPase-dependent polar growth. Plant Cell 18: 366-381,2006.
[64]Pasqualato S, Renault L, Cherfils J. Arf, Ar1, Arp and Sar proteins:a family of GTPbinding proteins with a structural device for "front-back" communication. EMBO Rep 3: 1035-1041,2002.
[65]Amor JC, Harrison DH, Kahn RA, et al. Structure of the human ADP-ribosylation factor 1 complexed with GDP. Nature 372:704-708,1994.
[66]Chardin P, Paris S, Antonny B, et al. A human exchange factor for ARF contains Sec7-and pleckstrin-homology domains. Nature 384:481-484,1996.
[67]Cherfils J, Menetrey J, Mathieu M, et al. Structure of the Sec7 domain of the Arf exchange factor ARNO. Nature 392:101-105,1998.
[68]Cherfils J, Melancon P. On the action of Brefeldin A on Sec7-stimulated membranerecruitment and GDP/GTP exchange of Arf proteins. Biochem Soc Trans 33: 635-638,2005.
[69]Beraud-Dufour S, Robineau S, Chardin P, et al. A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1. EMBO J 17:3651-3659,1998.
[70]Chardin P, Callebaut I. The yeast Sar exchange factor Sec 12, its higher organism orthologs, fold as beta-propellers. FEBS Lett 525:171-173,2002.
[71]Wu F, Liu Y, Zhu Z, et al. The 1.9 A crystal structure of Prp20p from Saccharomyces cerevisiae and its binding properties to Gsplp and histones. J Struct Biol 174:213-222, 2011.
[72]Delprato A, Lambright DG. Structural basis for Rab GTPase activation by VPS9 domain exchange factors. Nat Struct Mol Biol 14:406-412,2007.
[73]Delprato A, Merithew E, Lambright DG Structure, exchange determinants, and family-wide rab specificity of the tandem helical bundle and Vps9 domains of Rabex-5. Cell 118:607-617,2004.
[74]Sato Y, Fukai S, Ishitani R, et al. Crystal structure of the Sec4p-Sec2p complex in the nucleotide exchanging intermediate state. Proc Natl Acad Sci USA 104:8305-8310,2007.
[75]Itzen A, Rak A, Goody RS. Sec2 is a highly efficient exchange factor for the Rab protein Sec4. J Mol Biol 365:1359-1367,2007.
[76]Kim YG, Raunser S, Munger C, et al. The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell 127:817-830,2006.
[77]Renault L, Nassar N, Vetter I, et al. The 1.7 A crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller. Nature 392:97-101, 1998.
[78]Renault L, Kuhlmann J, Henkel A, et al. Structural basis for guanine nucleotide exchange on Ran by the regulator of chromosome condensation (RCC1). Cell 105:245-255,2001.
[79]Stewart M, Kent HM, McCoy AJ. The structure of the Q69L mutant of GDP-Ran shows a major conformational change in the switch II loop that accounts for its failure to bind nuclear transport factor 2 (NTF2). J Mol Biol 284:1517-1527,1998.
[80]De Rooij J, Zwartkruis FJ, Verheijen MH, et al. Epac is a Rapl guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474-477, 1998.
[81]Kawasaki H, Springett GM, Mochizuki N, et al. A family of cAMP-binding proteins that directly activate Rap1.Science 282:2275-2279,1998.
[82]De Rooij J, Rehmann H, van Triest M, et al. Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs. J Biol Chem 275:20829-20836,2000.
[83]Rehmann H, Das J, Knipscheer P, et al. Structure of the cyclic-AMPresponsive exchange factor Epac2 in its auto-inhibited state. Nature 439:625-628,2006.
[84]Cierpicki T, Bielnicki J, Zheng M, et al. The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA. Protein Sci 18:2067-2079,2009.
[85]Chhatriwala MK, Betts L, Worthylake DK, et al. The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation. J Mol Biol 368:1307-1320, 2007.
[86]Kristelly R, Gao G, Tesmer JJ. Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor. J Biol Chem 279:47352-47362,2004.
[87]Gureasko J, Kuchment O, Makino DL, et al. Role of the histone domain in the autoinhibition and activation of the Ras activator Son of Sevenless. Proc Natl Acad Sci USA 107:3430-3435,2010.
[88]Sondermann H, Soisson SM, Boykevisch S, et al. Structural analysis of autoinhibition in the Ras activator Son of sevenless. Cell 119:393-405,2004.
[89]Nimnual AS, Yatsula BA, Bar-Sagi D. Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. Science 279:560-563,1998.
[90]Shankaranarayanan A, Boguth CA, Lutz S, et al. Galpha q allosterically activates and relieves autoinhibition of p63RhoGEF. Cell Signal 22:1114-1123,2010.
[91]Lutz S, Shankaranarayanan A, Coco C, et al. Structure of Galphaq-p63RhoGEF-RhoA complex reveals a pathway for the activation of RhoA by GPCRs. Science 318:1923-1927, 2007.
[92]Derewenda U, Oleksy A, Stevenson AS, et al. The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca2τ sensitization pathway in smooth muscle. Structure 12:1955-1965,2004.
[93]Rossman KL, Worthylake DK, Snyder JT, et al. A crystallographic view of interactions between Dbs and Cdc42:PH domain-assisted guanine nucleotide exchange. EMBO J 21: 1315-1326,2002.
[94]Bellanger JM, Estrach S, Schmidt S, et al. Different regulation of the Trio Dbl-Homology domains by their associated PH domains. Biol Cell 95:625-634,2003.
[95]Baumeister MA, Rossman KL, Sondek J, et al. The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotideexchange activity. Biochem J 400:563-572,2006.
[96]Mitin N, Betts L, Yohe ME, et al. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nat Struct Mol Biol 14:814-823,2007.
[97]Murayama K, Shirouzu M, Kawasaki Y, et al. Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism. J Biol Chem 282:4238-4242,2007.
[98]Kawasaki Y, Senda T, Ishidate T, et al. Asef, a link between the tumor suppressor APC and G-protein signaling. Science 289:1194-1197,2000.
[99]Zhang Z, Chen L, Gao L, et al. Structural basis for the recognition of Asef by adenomatous polyposis coli. Cell Res 2011.
[100]Tsyba L, Nikolaienko O, Dergai O, et al. Intersectin multidomain adaptor proteins: regulation of functional diversity. Gene 473:67-75,2011.
[101]Ahmad KF, Lim WA. The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin. PLoS One 5:e11291,2010.
[102]Hussain NK, Jenna S, Glogauer M, et al. Endocytic protein intersectin-1 regulates actin assembly via Cdc42 and N-WASP. Nat Cell Biol 3:927-932,2001.
[103]Kintscher C, Wuertenberger S, Eylenstein R, et al. Autoinhibition of GEF activity in Intersectin 1 is mediated by the short SH3-DH domain linker. Protein Sci 19:2164-2174, 2010.
[104]Vigil D, Cherfils J, Rossman KL, et al. Ras superfamily GEFs and GAPs:validated and tractable targets for cancer therapy? Nat Rev Cancer 10:842-857,2010.
[105]Llorca O, Arias-Palomo E, Zugaza JL, et al. Global conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3. EMBO J 2005.
[106]Yu B, Martins IR, Li P, et al. Structural and energetic mechanisms of cooperative autoinhibition and activation of Vavl. Cell 140:246-256,2010.
[107]Chrencik JE, Brooun A, Zhang H, et al. Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vavl. J Mol Biol 380:828-843,2008.
[108]Rapley J, Tybulewicz VL, Rittinger K. Crucial structural role for the PH and C1 domains of the Vavl exchange factor. EMBO Rep 9:655-661,2008.
[109]Li P, Martins IR, Amarasinghe GK, et al. Internal dynamics control activation and activity of the autoinhibited Vav DH domain. Nat Struct Mol Biol 15:613-618,2008.
[110]Yohe ME, Rossman KL, Gardner OS, et al. Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif. J Biol Chem 282:13813-13823,2007.
[111]Yohe ME, Rossman K, Sondek J. Role of the C-terminal SH3 domain and N-terminal tyrosine phosphorylation in regulation of Tim and related Dbl-family proteins. Biochemistry 47:6827-6839,2008.
[112]Sternweis PC, Carter AM, Chen Z, et al. Regulation of Rho guanine nucleotide exchange factors by G proteins. Adv Protein Chem 74:189-228,2007.
[113]Jackson LP, Kelly BT, McCoy AJ, et al. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex. Cell 141: 1220-1229,2010.
[114]Yamaguchi K, Imai K, Akamatsu A, et al. SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice. Plant J 2011.
[115]Chen Z, Guo L, Hadas J, et al. Activation of p115-RhoGEF requires direct association of Galphal3 and the Dbl homology domain. J Biol Chem 287:25490-25500,2012.
[116]Lu M, Kinchen JM, Rossman KL, et al. PH domain of ELMO functions in trans to regulate Rac activation via Dock180. Nat Struct Mol Biol 11:756-762,2004.
[117]Lu M, Kinchen JM, Rossman KL, et al. A Steric-inhibition model for regulation of nucleotide exchange via the Dock180 family of GEFs. Curr Biol 15:371-377,2005.
[118]Hanawa-Suetsugu K, Kukimoto-Niino M, et al. Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms. Proc Natl Acad Sci USA 109:3305-3310,2012.
[119]Chabre M. Aluminofluoride and beryllofluoride complexes:a new phosphate analogs in enzymology. Trends Biochem Sci 15:6-10,1990.
[120]Mittal R, Ahmadian MR, Goody RS, et al. Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPaseactivating proteins. Science 273:115-117,1996.
[121]Wittinghofer A. Signaling mechanistics:aluminum fluoride for molecule of the year. Curr Biol 7:R682-685,1997.
[122]Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex:structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277:333-338,1997.
[123]Rittinger K, Walker PA, Eccleston JF, et al. Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue.Nature 389: 758-762,1997.
[124]Calmels TP, Callebaut I, Leger I, et al. Sequence and 3D structural relationships between mammalian Ras- and Rho-specific GTPase activating proteins (GAPs):the cradle fold. FEBS Lett 426:205-211,1998.
[125]Kurella VB, Richard JM, Parke CL, et al. Crystal structure of the GTPase-activating protein-related domain from IQGAP1. J Biol Chem 284:14857-14865,2009.
[126]Erdmann KS, Mao Y, McCrea HJ, et al. A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway. Dev Cell 13:377-390,2007.
[127]Pirruccello M, Swan LE, Folta-Stogniew E, et al. Recognition of the F&H motif by the Lowe syndrome protein OCRL. Nat Struct Mol Biol 18:789-795,2011.
[128]Raaijmakers JH, Bos JL. Specificity in Ras and Rap signaling. J Biol Chem 284: 10995-10999,2009.
[129]Raaijmakers JH, Bos JL. Specificity in Ras and Rap signaling. J Biol Chem 284: 10995-10999,2009.
[130]Bernards A. GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochim Biophys Acta 1603:47-82,2003.
[131]Kupzig S, Bouyoucef-Cherchalli D, Yarwood S, et al. The ability of GAP1IP4BP to function as a Rap1 GTPase-activating protein (GAP) requires its Ras GAP-related domain and an arginine finger rather than an asparagine thumb. Mol Cell Biol 29:3929-3940, 2009.
[132]Pena V, Hothorn M, Eberth A, et al. The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction. EMBO Rep 9:350-355,2008.
[133]Sot B, Kotting C, Deaconescu D, et al. Unravelling the mechanism of dual-specificity GAPs. EMBO J 29:1205-1214,2010.
[134]Daumke O, Weyand M, Chakrabarti PP, et al. The GTPaseactivating protein Rapl GAP uses a catalytic asparagine. Nature 429:197-201,2004.
[135]Scrima A, Thomas C, Deaconescu D, et al. The Rap-RapGAP complex:GTP hydrolysis without catalytic glutamine and arginine residues. EMBO J 27:1145-1153,2008.
[136]Menetrey J, Cherfils J. Structure of the smallGprotein Rap2 in a non-catalytic complex with GTP. Proteins 37:465-473,1999.
[137]Strom M, Vollmer P, Tan TJ, et al. A yeast GTPase-activating protein that interacts specifically with a member of the Ypt/Rab family. Nature 361:736-739,1993.
[138]Rak A, Fedorov R, Alexandrov K, et al. Crystal structure of the GAP domain of Gyplp: first insights into interaction with Ypt/Rab proteins. EMBO J 19:5105-5113,2000.
[139]Tempel W, Tong Y, Dimov S, et al. First crystallographic models of human TBC domains in the context of a family-wide structural analysis. Proteins 71:497-502,2008.
[140]Pan X, Eathiraj S, Munson M, et al. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442:303-306,2006.
[141]Nottingham RM, Ganley IG, Barr FA, et al. RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins. J Biol Chem 286:33213-33222, 2011.
[142]Ismail SA, Vetter IR, Sot B, et al. The structure of an Arf-ArfGAP complex reveals a Ca2 r regulatory mechanism. Cell 141:812-821,2010.
[143]Veltel S, Kravchenko A, Ismail S, et al. Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3-effector-GAP complex. FEBS Lett 582:2501-2507,2008.
[144]Miller EA, Barlowe C. Regulation of coat assembly:sorting things out at the ER. Curr Opin Cell Biol 22:447-453,2010.
[145]Antonny B, Madden D, Hamamoto S, et al. Dynamics of the COPII coat with GTP and stable analogues. Nat Cell Biol 3:531-537,2001.
[146]Bi X, Mancias JD, Goldberg J. Insights into COPII coat nucleation from the structure of Sec23. Sar1 complexed with the active fragment of Sec31. Dev Cell 13:635-645,2007.
[147]Bi X, Corpina RA, Goldberg J. Structure of the Sec23/24-Sarl pre-budding complex of the COPII vesicle coat. Nature 419:271-277,2002.
[148]Karnoub AE, Weinberg RA. Ras oncogenes:split personalities. Nat Rev Mol Cell Biol 9:517-531,2008.
[149]Canagarajah B, Leskow FC, Ho JY, et al. Structural mechanism for lipid activation of the Rac-specific GAP, beta2-chimaerin. Cell 119:407-418,2004.
[150]Bigay J, Gounon P, Robineau S, et al. Lipid packing sensed by ArfGAPl couples COPI coat disassembly to membrane bilayer curvature. Nature 426:563-566,2003.
[151]Bankaitis VA, Mousley CJ, Schaaf G The Sec14 superfamily and mechanisms for crosstalk between lipid metabolism and lipid signaling. Trends Biochem Sci 35:150-160,2010.
[152]Sirokmany G, Szidonya L, Kaldi K, et al. Sec14 homology domain targets p50RhoGAP to endosomes and provides a link between Rab and Rho GTPases. J Biol Chem 281: 6096-6105,2006.
[153]Moskwa P, Paclet MH, Dagher MC, et al. Autoinhibition of p50 Rho GTPaseactivating protein (GAP) is released by prenylated small GTPases. J Biol Chem 280:6716-6720, 2005.
[154]Zhou YT, Chew LL, Lin SC, et al. The BNIP-2 and Cdc42GAP homology (BCH) domain of p50RhoGAP/Cdc42GAP sequesters RhoA from inactivation by the adjacent GTPase-activating protein domain. Mol Biol Cell 21:3232-3246,2010.
[155]Eberth A, Lundmark R, Gremer L, et al. A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family. Biochem J 417:371-377,2009.
[156]Peter BJ, Kent HM, Mills IG, et al. BAR domains as sensors of membrane curvature:the amphiphysin BAR structure. Science 303:495-499,2004.
[157]Fauchereau F, Herbrand U, Chafey P, et al. The RhoGAP activity of OPHN1, a new F-actin-binding protein, is negatively controlled by its amino-terminal domain. Mol Cell Neurosci 23:574-586,2003.
[158]Drin G, Morello V, Casella JF, et al. Asymmetric tethering of flat and curved lipid membranes by a golgin. Science 320:670-673,2008.
[159]Jian X, Brown P, Schuck P, et al. Autoinhibition of Arf GTPase-activating protein activity by the BAR domain in ASAP1. J Biol Chem 284:1652-1663,2009.
[160]Tong Y, Tempel W, Wang H, et al. Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin alpha1. Proc Natl Acad Sci USA 107:20346-20351,2010.
[161]Scheffzek K, Ahmadian MR, Wiesmuller L, et al. Structural analysis of the GAP-related domain from neurofibromin and its implications. EMBO J 17:4313-4327,1998.
[162]D'Angelo I, Welti S, Bonneau F, et al. A novel bipartite phospholipid-binding module in the neurofibromatosis type 1 protein. EMBO Rep 7:174-179,2006.
[163]Welti S, Fraterman S, D'Angelo I, et al. The sec 14 homology module of neurofibromin binds cellular glycerophospholipids:mass spectrometry and structure of a lipid complex. J Mol Biol 366:551-562,2007.
[164]He H, Yang T, Terman JR, et al. Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration. Proc Natl Acad Sci USA 106:15610-15615,2009.
[165]Bell CH, Aricescu AR, Jones EY, et al. A dual binding mode for RhoGTPases in plexin signalling. PLoS Biol 9:e1001134,2011.
[166]Margarit SM, Sondermann H, Hall BE, et al. Structural evidence for feedback activation by Ras GTP of the Ras-specific nucleotide exchange factor SOS. Cell 112:685-695,2003.
[167]Gureasko J, Galush WJ, Boykevisch S, et al. Membrane-dependent signal integration by the Ras activator Son of sevenless. Nat Struct Mol Biol 15:452-461,2008.
[168]Roose JP, Mollenauer M, Ho M, et al. Unusual interplay of two types of Ras activators, RasGRP and SOS, establishes sensitive and robust Ras activation in lymphocytes. Mol Cell Biol 27:2732-2745,2007.
[169]Innocenti M, Tenca P, Frittoli E, et al. Mechanisms through which Sos-1 coordinates the activation of Ras and Rac. J Cell Biol 156:125-136,2002.
[170]Scita G, Nordstrom J, Carbone R, et al. EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401:290-293,1999.
[171]Wang W, Fisher EM, Jia Q, et al. The Grb2 binding domain of mSosl is not required for downstream signal transduction. Nat Genet 10:294-300,1995.
[172]DiNitto JP, Delprato A, Gabe Lee MT, et al. Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors. Mol Cell 28:569-583,2007.
[173]Cohen LA, Honda A, Varnai P, et al. Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains. Mol Biol Cell 18:2244-2253,2007.
[174]Stalder D, Barelli H, Gautier R, et al. Kinetic studies of the Arf activator Arno on model membranes in the presence of Arf effectors suggest control by a positive feedback loop. J Biol Chem 286:3873-3883,2011.
[175]Cronin TC, DiNitto JP, Czech MP, et al. Structural determinants of phosphoinositide selectivity in splice variants of Grpl family PH domains. EMBO J 23:3711-3720,2004.
[176]Menetrey J, Perderiset M, Cicolari J, et al. Structural basis for ARF1-mediated recruitment of ARHGAP21 to Golgi membranes. EMBO J 2007.
[177]Nemergut ME, Mizzen CA, Stukenberg T, et al. Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2B. Science 292:1540-1543,2001.
[178]Makde RD, England JR, Yennawar HP, et al. Structure of RCC1 chromatin factor bound to the nucleosome core particle. Nature 467:562-566,2010.
[179]Koyama M, Matsuura Y. An allosteric mechanism to displace nuclear export cargo from CRM1 and RanGTP by RanBP1. EMBO J 29:2002-2013,2010.
[180]Seewald MJ, Korner C, Wittinghofer A, et al. RanGAP mediates GTP hydrolysis without an arginine finger. Nature 415:662-666,2002.
[181]Nemergut ME, Lindsay ME, Brownawell AM, et al. Ran-binding protein 3 links Crml to the Ran guanine nucleotide exchange factor. J Biol Chem 277:17385-17388,2002.
[182]Langer K, Dian C, Rybin V, et al. Insights into the function of the CRM1 cofactor RanBP3 from the structure of its Ran-binding domain. PLoS One 6:e17011,2011.
[183]Rivera-Molina FE, Novick PJ. A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway. Proc Natl Acad Sci USA 106:14408-14413,2009.
[184]Rink J, Ghigo E, Kalaidzidis Y, et al. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122:735-749,2005.
[185]Horiuchi H, Lippe R, McBride HM, et al. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90:1149-1159,1997.
[186]Zhu H, Zhu G, Liu J, et al. Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell. Mol Biol Cell 18:4119-4128,2007.
[187]Lippe R, Miaczynska M, Rybin V, et al. Functional synergy between Rab5 effector Rabaptin-5 and exchange factor Rabex-5 when physically associated in a complex. Mol Biol Cell 12:2219-2228,2001.
[188]Medkova M, France YE, Coleman J, et al. The rab exchange factor Sec2p reversibly associates with the exocyst. Mol Biol Cell 17:2757-2769,2006.
[189]Mizuno-Yamasaki E, Medkova M, Coleman J, et al. Phosphatidylinositol 4-phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF Sec2p. Dev Cell 18:828-840,2010.
[190]Bustelo XR, Sauzeau V, Berenjeno IM. GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo. Bioessays 29:356-370,2007.
[191]Hoelz A, Janz JM, Lawrie SD, et al. Crystal structure of the SH3 domain of betaPIX in complex with a high affinity peptide from PAK2. J Mol Biol 358:509-522,2006.
[192]Manser E, Loo TH, Koh CG, et al. PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol Cell 1:183-192,1998.
[193]Lei M, Lu W, Meng W, et al. Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch. Cell 102:387-397,2000.
[194]Parrini MC, Lei M, Harrison SC, et al. Pakl kinase homodimers are autoinhibited in trans and dissociated upon activation by Cdc42 and Racl. Mol Cell 9:73-83,2002.
[195]Li Z, Hannigan M, Mo Z, et al. Directional sensing requires G beta gamma-mediated PAK1 and PIX alphadependent activation of Cdc42. Cell 114:215-227,2003.
[196]Takaku T, Ogura K, Kumeta H, et al. Solution structure of a novel Cdc42 binding module of Beml and its interaction with Ste20 and Cdc42. J Biol Chem 285:19346-19353,2010.
[197]Butty AC, Perrinjaquet N, Petit A, et al. A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization. EMBO J 21:1565-1576, 2002.
[198]Kozubowski L, Saito K, Johnson JM, et al. Symmetry-breaking polarization driven by a Cdc42p GEF-PAK complex. Current Biol 18:1719-1726,2008.
[199]Connolly BA, Rice J, Feig LA, et al. Tiaml-IRSp53 complex formation directs specificity of rac-mediated actin cytoskeleton regulation. Mol Cell Biol 25:4602-4614,2005.
[200]Rajagopal S, Ji Y, Xu K, et al. Scaffold proteins IRSp53 and spinophilin regulate localized Rac activation by T-lymphocyte invasion and metastasis protein 1 (TIAM1). J Biol Chem 285:18060-18071,2010.
[201]Nishimura T, Yamaguchi T, Kato K, et al. PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1. Nat Cell Biol 7:270-277,2005.
[202]Ten Klooster JP, Evers EE, Janssen L, et al. Interaction between Tiaml and the Arp2/3 complex links activation of Rac to actin polymerization. Biochem J 397:39-45,2006.
[203]Jenna S, Hussain NK, Danek El, et al.The activity of the GTPase-activating protein CdGAP is regulated by the endocytic protein intersectin. J Biol Chem 277:6366-6373, 2002.
[204]Primeau M, Ben Djoudi Ouadda A, Lamarche-Vane N. Cdc42 GTPase-activating protein (CdGAP) interacts with the SH3D domain of Intersectin through a novel basic-rich motif. FEBS Lett 585:847-853,2011.
[205]Zhao X, Lasell TK, Melancon P. Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments:evidence for distinct functions in protein traffic. Mol Biol Cell 13:119-133,2002.
[206]Deng Y, Golinelli-Cohen MP, Smirnova E, et al. A COPI coat subunit interacts directly with an early-Golgi localized Arf exchange factor. EMBO Rep 10:58-64,2009.
[207]Lefrancois S, McCormick PJ. The Arf GEF GBF1 is required for GGA recruitment to Golgi membranes. Traffic 8:1440-1451,2007.
[208]Neunuebel MR, Chen Y, Gaspar AH, et al. De-AMPylation of the small GTPase Rabl by the pathogen Legionella pneumophila. Science 333:453-456,2011.
[209]Hanzal-Bayer M, Renault L, Roversi P, et al. The complex of Ar12-GTP and PDE delta: from structure to function. EMBO J 21:2095-2106,2002.
[210]Yu X, Breitman M, Goldberg J. A structure-based mechanism for Arf1-dependent recruitment of coatomer to membranes. Cell 148:530-542,2012.
[211]Beck R, Rawet M, Wieland FT, et al. The COPI system:molecular mechanisms and function. FEBS Lett 583:2701-2709,2009.
[212]Hsu VW, Lee SY, Yang JS. The evolving understanding of COPI vesicle formation. Nat Rev Mol Cell Biol 10:360-364,2009.
[213]Lee SY, Yang JS, Hong W, et al. ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation. J Cell Biol 168:281-290,2005.
[214]Reinhard C, Schweikert M, Wieland FT, et al. Functional reconstitution of COPI coat assembly and disassembly using chemically defined components. Proc Natl Acad Sci USA 100:8253-8257,2003.
[215]Ismail SA, Chen YX, Rusinova A, et al. Arl2-GTP and Arl3-GTP regulate a GDIlike transport system for farnesylated cargo. Nat Chem Biol 2011.
[216]Bai M, Gad H, Turacchio G, et al. ARFGAP1 promotes AP-2-dependent endocytosis. Nat Cell Biol 13:559-567,2011.
[217]Bischoff FR, Krebber H, Smirnova E, et al. Co-activation of Ran-GTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1. EMBO J 14:705-715,1995.
[218]Ohlson MB, Huang Z, Alto NM, et al. Structure and function of Salmonella SifA indicate that its interactions with SKIP, SseJ, and RhoA family GTPases induce endosomal tubulation. Cell Host Microbe 4:434-446,2008.
[219]Loirand G, Pacaud P. The role of Rho protein signaling in hypertension. Nat Rev Cardiol 7: 637-647,2010.
[220]Aktories K. Bacterial protein toxins that modify host regulatory GTPases. Nat Rev Microbiol 9:487-498,2011.
[221]Newey SE, Velamoor V, Govek EE, et al. Rho GTPases, dendritic structure,and mental retardation. J Neurobiol 64:58-74,2005.
[222]Tidyman WE, Rauen KA. The RASopathies:developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 19:230-236,2009.
[223]Klose A, Ahmadian MR, Schuelke M, et al. Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1. Hum Mol Genet 7:1261-1268,1998.
[224]Kuhnel K, Veltel S, Schlichting I, et al. Crystal structure of the human retinitis pigmentosa 2 protein and its interaction with Ar13. Structure 14:367-378,2006.
[225]Mayer K, Goedbloed M, van Zijl K, et al. Characterisation of a novel TSC2 missense mutation in the GAP related domain associated with minimal clinical manifestations of tuberous sclerosis. J Med Genet 41:e64,2004.
[226]D'Adamo P, Menegon A, Lo Nigro C, et al. Mutations in GDI1 are responsible for X-linked non-specific mental retardation. Nat Genet 19:134-139,1998.
[227]Rak A, Pylypenko O, Durek T, et al. Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase. Science 302:646-650,2003.
[228]Lepri F, De Luca A, Stella L, et al. SOS1 mutations in Noonan syndrome:molecular spectrum, structural insights on pathogenic effects, and genotype-phenotype correlations. Hum Mutat 32:760-772,2011.
[229]Roberts AE, Araki T, Swanson KD, et al. Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 39:70-74,2007.
[230]Tartaglia M, Pennacchio LA, Zhao C, et al. Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 39:75-79,2007.
[231]Southgate L, Machado RD, Snape KM, et al. Gain-of-function mutations of ARHGAP31, a Cdc42/Rac1 GTPase regulator, cause syndromic cutis aplasia and limb anomalies. Am J Hum Genet 88:574-585,2011.
[232]Shaheen R, Faqeih E, Sunker A, et al. Recessive mutations in DOCK6, encoding the guanidine nucleotide exchange factor DOCK6, lead to abnormal actin cytoskeleton organization and Adams-Oliver syndrome. Am J Hum Genet 89:328-333,2011.
[233]Orrico A, Galli L, Faivre L, et al. Aarskog-Scott syndrome:clinical update and report of nine novel mutations of the FGD1 gene. Am J Med Genet 152A:313-318,2010.
[234]Nystrom AM, Ekvall S, Allanson J, et al. Noonan syndrome and neurofibromatosis type I in a family with a novel mutation in NF1. Clin Genet 76:524-534,2009.
[235]Maheshwar MM, Cheadle JP, Jones AC, et al.The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Hum Mol Genet 6:1991-1996,1997.
[236]Nellist M, Sancak O, Goedbloed MA, et al. Distinct effects of single amino-acid changes to tuberin on the function of the tuberin-hamartin complex. Eur J Hum Genet 13:59-68,2005.
[237]Falace A, Filipello F, La Padula V, et al. TBC1D24, an ARF6-interacting protein, is mutated in familial infantile myoclonic epilepsy. Am J Hum Genet 87:365-370,2010.
[238]Shoubridge C, Tarpey PS, Abidi F, et al. Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability. Nat Genet 42:486-488, 2010.
[239]Shoubridge C, Walikonis RS, Gecz J, et al. Subtle functional defects in the Arfspecific guanine nucleotide exchange factor IQSEC2 cause non-syndromic X-linked intellectual disability. Small GTPases 1:98-103,2010.
[240]Kutsche K, Yntema H, Brandt A, et al. Mutations in ARHGEF6, encoding a guanine nucleotide exchange factor for Rho GTPases, in patients with X-linked mental retardation. Nat Genet 26:247-250,2000.
[241]Billuart P, Bienvenu T, Ronce N, et al. Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation. Nature 392:923-926,1998.
[242]Hamdan FF, Gauthier J, Spiegelman D, et al. Mutations in SYNGAP1 in autosomal nonsyndromic mental retardation. N Engl J Med 360:599-605,2009.
[243]Gedeon AK, Colley A, Jamieson R, et al. Identification of the gene (SEDL) causing X-linked spondyloepiphyseal dysplasia tarda. Nat Genet 22:400-404,1999.
[244]Zong M, Wu XG, Chan CW, et al. The adaptor function of TRAPPC2 in mammalian TRAPPs explains TRAPPC2-associated SEDT and TRAPPC9-associated congenital intellectual disability. PLoS One 6:e23350,2011.
[245]Sheen VL, Ganesh VS, Topcu M, et al. Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex. Nat Genet 36:69-76,2004.
[246]Canagarajah B, Leskow FC, Ho JY, et al. Structural mechanism for lipid activation of the Rac-specific GAP, beta2-chimaerin. Cell 119:407-418,2004.
[247]Pommier Y, Cherfils J. Interfacial inhibition of macromolecular interactions:nature's paradigm for drug discovery. Trends Pharmacol Sci 26:138-145,2005.
[248]Zeghouf M, Guibert B, Zeeh JC, et al. Arf, Sec7 and Brefeldin A:a model towards the therapeutic inhibition of guanine nucleotide-exchange factors. Biochem Soc Trans 33: 1265-1268,2005.