Biochemical characterization of yeast RAS2 mutants reveals a new region of ras protein involved in the interaction with GTPase-activating proteins.
Biochemical characterization of yeast RAS2 mutants reveals a new region of ras protein involved in the interaction with GTPase-activating proteins.
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DOI：
10.1016/s0021-9258(17)37690-1
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发表时间：
1994-02
期刊：
影响因子：
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通讯作者：
D. Wood;P. Poullet;B. Wilson;M. Khalil;K. Tanaka;J. Cannon;F. Tamanoi
中科院分区：
文献类型：
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作者：
D. Wood;P. Poullet;B. Wilson;M. Khalil;K. Tanaka;J. Cannon;F. Tamanoi
我们报道了最近发现的酵母RAS2的两个突变体RAS2-E99K和RAS2-E130K的生化特征。这些突变体在酵母中表现出显性的激活表型。对RAS2-E99K突变体蛋白的GTP酶活性、GDP解离活性以及激活腺苷环化酶活性的研究表明，RAS2-E99K突变体蛋白的这些活性与野生型蛋白相似。另一方面，RAS2-E130K蛋白与野生型蛋白不同，它的GDP解离速度快，腺苷环化酶的活性高2倍。当检测对GTP酶激活蛋白(GAP)的敏感性时，我们发现RAS2-E99K蛋白对NF1-GAP活性的敏感性大约降低1200倍。此外，竞争结合实验表明，RAS2-E99K蛋白与NF1的亲和力降低了150多倍。因此，RAS2-E99K突变影响与GAP蛋白的相互作用。这种突变特别有趣，因为它是在ras蛋白的阿尔法3区发现的第一个影响间隙相互作用的突变。α3区似乎直接参与了与NF1的相互作用，因为包含RAS2残基99的序列的多肽抑制了NF1-GAP的活性。这些结果表明，RAS和GAP之间的相互作用涉及RAS内比先前认识到的更大的区域。
We report biochemical characterization of two recently identified mutants of yeast RAS2, RAS2-E99K and RAS2-E130K. These mutants exhibit dominant activating phenotypes in yeast. Characterization of their intrinsic GTPase and GDP dissociation as well as their ability to stimulate adenylate cyclase showed that these activities of RAS2-E99K mutant protein were similar to those of the wild type protein. RAS2-E130K protein, on the other hand, differed from the wild type protein with a fast GDP dissociation rate and 2-fold higher activation of adenylate cyclase. When the sensitivity to GTPase-activating protein (GAP) was examined, we found that the RAS2-E99K protein was approximately 1200-fold less sensitive to NF1-GAP activity. In addition, the affinity for NF1 as revealed by competition binding experiments was reduced more than 150-fold with RAS2-E99K protein. Thus, the RAS2-E99K mutation affects interaction with GAP proteins. This mutation is particularly interesting because it is the first mutation identified in the alpha 3 region of ras protein that affects GAP interaction. The alpha 3 region appears to be directly involved in interaction with NF1, since peptides containing the sequence encompassing residue 99 of RAS2 inhibit NF1-GAP activity. These results suggest that the interaction between ras and GAP involves a larger region within ras than previously recognized.