Nonaka H, Tanaka K, Hirano H, Fujiwara T, Kohno H, Umikawa M, Mino A, Takai Y
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Japan.
EMBO J. 1995 Dec 1;14(23):5931-8. doi: 10.1002/j.1460-2075.1995.tb00281.x.
The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.
酿酒酵母中的RHO1基因编码哺乳动物RhoA小GTP结合蛋白的同源物，该蛋白与各种肌动蛋白细胞骨架依赖性细胞功能有关。在酵母中，Rho1p参与芽的形成。用RhoA取代RHO1的酵母菌株表现出隐性温度敏感生长表型。从该菌株中分离出一个显性抑制突变体。抑制基因的分子克隆表明，该突变发生在PKC1的假底物位点，PKC1是哺乳动物蛋白激酶C的酵母同源物。双杂交分析表明，GTP-Rho1p而非GDP-Rho1p与含有假底物位点和C1结构域的Pkc1p区域相互作用。MKK1和MPK1分别编码促分裂原活化蛋白激酶激酶和促分裂原活化蛋白激酶同源物，并在PKC1的下游发挥作用。显性活性MKK1-6突变或MPK1的过表达抑制了RhoA突变体的温度敏感性。PKC1的显性激活突变抑制了RhoA突变体的温度敏感性。PKC1的显性激活突变抑制了RHO1的两个效应突变体rho1(F44Y)和rho1(E451)的温度敏感性，但不抑制rho1(V43T)的温度敏感性。这些结果表明，至少有两条由Rho1p调节的信号通路，其中一条下游靶点是Pkc1p，导致促分裂原活化蛋白激酶级联反应的激活。
