Tognon C E, Kirk H E, Passmore L A, Whitehead I P, Der C J, Kay R J
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada V5Z 4E6.
Mol Cell Biol. 1998 Dec;18(12):6995-7008. doi: 10.1128/MCB.18.12.6995.
As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.
作为对诱导NIH 3T3成纤维细胞转化的克隆进行cDNA文库筛选的一部分，我们分离出了一个编码鸟嘌呤核苷酸交换因子RasGRP小鼠同源物的cDNA。一个预计会阻止与Ras相互作用的点突变消除了小鼠RasGRP（mRasGRP）转化成纤维细胞和激活丝裂原活化蛋白激酶（MAP激酶）的能力。通过共表达H-Ras、K-Ras和N-Ras可增强经由mRasGRP的MAP激酶激活，而通过共表达H-Ras和K-Ras的显性负性形式可部分抑制这种激活。mRasGRP的C末端包含一对EF手结构和一个C1结构域，该结构域与蛋白激酶C的佛波酯和二酰基甘油结合C1结构域非常相似。删除EF手结构不会影响mRasGRP转化NIH 3T3细胞的能力。相反，删除C1结构域或相邻的碱性氨基酸簇会消除mRasGRP的转化活性。如果缺失的C1结构域被膜定位的异戊二烯化信号或蛋白激酶C的二酰基甘油和佛波酯结合C1结构域取代，经由mRasGRP的转化和MAP激酶激活得以恢复。当血清浓度较低时，佛波酯可调节mRasGRP的转化活性，且佛波酯的这种作用依赖于mRasGRP的C1结构域。C1结构域还可赋予K-Ras的异戊二烯化缺陷型突变体佛波醇肉豆蔻酸酯乙酸盐调节的转化活性。C1结构域介导mRasGRP响应佛波酯或血清刺激向细胞膜的转位。这些结果表明，成纤维细胞中mRasGRP激活的主要机制是通过其被募集到富含二酰基甘油的膜上。mRasGRP在淋巴组织、大脑以及一些淋巴样细胞系中表达。在这些细胞中，RasGRP有可能作为刺激产生二酰基甘油的磷脂酶C的受体与Ras激活之间的直接联系。
