Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras

被引:128
作者
Fam, NP
Fan, WT
Wang, ZX
Zhang, LJ
Chen, H
Moran, MF
机构
[1] UNIV TORONTO, BANTING & BEST DEPT MED RES, TORONTO, ON M5G 1L6, CANADA
[2] UNIV TORONTO, DEPT MED & MOL GENET, TORONTO, ON M5G 1L6, CANADA
关键词
D O I
10.1128/MCB.17.3.1396
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli, Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region, Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association,vith calmodulin in epithelial cells ectopically expressing RaS-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.
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页码:1396 / 1406
页数:11
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