Ras-Specific Exchange Factor GRF: Oligomerization through Its Dbl Homology Domain and Calcium-Dependent Activation of Raf
Ras-Specific Exchange Factor GRF: Oligomerization through Its Dbl Homology Domain and Calcium-Dependent Activation of Raf
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Ras 特异性交换因子 GRF：通过其 Dbl 同源域进行寡聚化和 Raf 的钙依赖性激活
DOI：
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发表时间：
1999
影响因子：
5.3
通讯作者：
D. Lowy
中科院分区：
文献类型：
--
作者：
P. H. Anborgh;X. Qian;A. Papageorge;W. Vass;J. Declue;D. Lowy
摘要RAS特异性交换因子RAS-GRF1(GRF1)和RAS-GRF2(GRF2)的全长版本在脑和有限数量的其他器官中表达，在293T细胞中具有离子依赖的ERK丝裂原激活蛋白激酶活性(C.L.Farnsworth等，Nature 376：524-527,1995；N.P.Fam等，Mol)。牢房。比奥尔。17：1396-1406,1996)。每个GRF蛋白都含有一个DBL同源(DH)结构域。用酵母双杂交技术筛选与GRF1的水解区相关的多肽。在这个筛选中，从人脑cDNA文库中分离到一个阳性克隆，该克隆由GRF2水解区及其邻近的苯丙喹酮结构域组成。缺失分析表明，双杂交相互作用只需要水解域，GRF1水解域N端的Leu-263突变为Gln(L263Q)取消了双杂交相互作用，而水解域中更多位于C末端的突变并没有消除相互作用。在大鼠脑提取液中检测到GRF1和GRF2之间的寡聚体，并在培养的哺乳动物细胞中强制表达GRF1和GRF2形成同源和异源寡聚体。在GRF1中引入L263Q突变导致蛋白质缺乏寡聚体形成，而含有DH簇突变的GRF1形成同源低聚物，其效率与野生型相似。与野生型GRF1相比，GRF1DH簇突变体对NIH3T3细胞的致灶活性降低，而L263Q突变体对NIH3T3细胞的凝集活性降低。这两个突变体在293T细胞中介导离子霉素依赖的ERK活性的能力都受到了损害。在没有离子霉素的情况下，表达野生型GRF1的293T细胞含有比对照细胞高得多的RAS-GTP；离子霉素诱导表达GRF1的细胞中ERK活性的增加也伴随着Raf激酶活性的增加，但没有进一步增加RAS-GTP的水平。我们的结论是，GRF1和GRF2可以通过它们的DH域形成同源和异质低聚物，GRF1对低聚物形成的突变失活与生物学和信号活性的受损有关，在293T细胞中，GRF1至少介导了两条激活Raf的途径：一条主要依赖RAS的构成信号，另一条是离子霉素诱导的信号，它与构成信号协同作用，而不进一步增加GTP-RAS的水平。
ABSTRACT The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2), which are expressed in brain and a restricted number of other organs, possess an ionomycin-dependent activation of Erk mitogen-activated protein kinase activity in 293T cells (C. L. Farnsworth et al., Nature 376:524–527, 1995; N. P. Fam et al., Mol. Cell. Biol. 17:1396–1406, 1996). Each GRF protein contains a Dbl homology (DH) domain. A yeast two-hybrid screen was used to identify polypeptides that associate with the DH domain of GRF1. In this screen, a positive cDNA clone from a human brain cDNA library was isolated which consisted of the GRF2 DH domain and its adjacent ilimaquinone domain. Deletion analysis verified that the two-hybrid interaction required only the DH domains, and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain abolished the two-hybrid interaction, while a cluster of more C-terminally located mutations in the DH domain did not eliminate the interaction. Oligomers between GRF1 and GRF2 were detected in a rat brain extract, and forced expression of GRF1 and GRF2 in cultured mammalian cells formed homo- and hetero-oligomers. Introduction of the L263Q mutation in GRF1 led to a protein that was deficient in oligomer formation, while GRF1 containing the DH cluster mutations formed homo-oligomers with an efficiency similar to that of wild type. Compared to wild-type GRF1, the focus-forming activity on NIH 3T3 cells of the GRF1 DH cluster mutant was reduced, while the L263Q mutant was inactive. Both mutants were impaired in their ability to mediate ionomycin-dependent Erk activity in 293T cells. In the absence of ionomycin, 293T cells expressing wild-type GRF1 contained much higher levels of Ras-GTP than control cells; the increase in Erk activity induced by ionomycin in the GRF1-expressing cells also induced a concomitant increase in Raf kinase activity, but without a further increase in the level Ras-GTP. We conclude that GRF1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oligomer formation by GRF1 is associated with impaired biological and signaling activities, and that in 293T cells GRF1 mediates at least two pathways for Raf activation: one a constitutive signal that is mainly Ras-dependent, and one an ionomycin-induced signal that cooperates with the constitutive signal without further augmenting the level of GTP-Ras.
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影响因子：
10.5
作者：
DURFEE, T;BECHERER, K;ELLEDGE, SJ
通讯作者：
ELLEDGE, SJ
影响因子：
4.4
作者：
MARCHUK, DA;SAULINO, AM;COLLINS, FS
通讯作者：
COLLINS, FS
影响因子：
3.5
作者：
Tonini, R;Franceschetti, S;Zippel, R
通讯作者：
Zippel, R
DOI：
10.1073/pnas.93.20.10803
发表时间：
1996-10-01
影响因子：
11.1
作者：
Enslen, H;Tokumitsu, H;Soderling, TR
通讯作者：
Soderling, TR