Itoh T, Kaibuchi K, Masuda T, Yamamoto T, Matsuura Y, Maeda A, Shimizu K, Takai Y
Department of Biochemistry, Kobe University School of Medicine, Japan.
Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):975-9. doi: 10.1073/pnas.90.3.975.
To identify the direct target molecule of ras p21 in higher eukaryotes, we have recently developed the cell-free system in which ras p21 activates mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK). In this cell-free system, the guanosine 5'-[gamma-thio]triphosphate- bound form of Ki-ras p21, but not the GDP-bound form, activates endogenous Xenopus MAP kinase as well as recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes. We separated two protein factors from the cytosol fraction of Xenopus oocytes by column chromatography: one was the inactive form of MAP kinase kinase and the other was a factor tentatively named ras p21-dependent ERK-kinase stimulator (REKS). The former and latter showed M(r) values of approximately 45,000 and 150,000-200,000, respectively, as estimated by gel filtration. Both factors were necessary for Ki-ras p21-dependent activation of MAP kinase/ERK2. These results indicate that an additional protein factor (REKS) is essential for Ki-ras p21 to activate MAP kinase through MAP kinase kinase.
为了鉴定高等真核生物中ras p21的直接靶分子，我们最近开发了一种无细胞系统，其中ras p21可激活丝裂原活化蛋白（MAP）激酶/细胞外信号调节激酶（ERK）。在这个无细胞系统中，在非洲爪蟾卵母细胞的胞质溶胶组分存在的情况下，鸟苷5'-[γ-硫代]三磷酸结合形式的Ki-ras p21（而非GDP结合形式）可激活内源性非洲爪蟾MAP激酶以及重组ERK2。我们通过柱色谱法从非洲爪蟾卵母细胞的胞质溶胶组分中分离出两种蛋白质因子：一种是MAP激酶激酶的无活性形式，另一种是暂命名为ras p21依赖性ERK激酶刺激物（REKS）的因子。通过凝胶过滤估计，前者和后者的相对分子质量分别约为45,000和150,000 - 200,000。这两种因子都是Ki-ras p21依赖性激活MAP激酶/ERK2所必需的。这些结果表明，一种额外的蛋白质因子（REKS）对于Ki-ras p21通过MAP激酶激酶激活MAP激酶至关重要。
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