Yamaguchi Yoshihiro, Ota Kazuhisa, Ito Takashi
Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561, Japan.
J Biol Chem. 2007 Jan 5;282(1):29-38. doi: 10.1074/jbc.M609308200. Epub 2006 Nov 7.
In Saccharomyces cerevisiae, the Rho-type small GTPase Cdc42 is activated by its guanine-nucleotide exchange factor Cdc24 to polarize the cell for budding and mating. A multidomain protein Bem1 interacts not only with Cdc42 but also with Cdc24 and the effectors of Cdc42, including the p21-activated kinase Ste20, to function as a scaffold for cell polarity establishment. Although Bem1 interacts with Cdc24 and Ste20 via its PB1 and the second SH3 domains (SH3b), respectively, it is unclear how Bem1 binds Cdc42. Here we show that a region comprising the SH3b and its C-terminal flanking segment termed CI (SH3b-CI) directly interacts with Cdc42. A dual-bait reverse two-hybrid approach revealed that the CI is critical to the interaction: N253D substitution in the CI abolishes the binding of the SH3b-CI to Cdc42 but not to the proline-rich region of Ste20, whereas W192K substitution in the SH3b has the opposite effect. Nevertheless, the SH3b-CI interacts with Ste20 proline-rich region and Cdc42 in a mutually exclusive manner. The N253D substitution renders cellular growth temperature-sensitive and suppresses mating. The W192K-induced mating defect is exacerbated by the N253D substitution and suppressed by increasing the dosage of Ste20 provided that the CI is intact. Intriguingly, Cdc42 can mediate an indirect interaction of the SH3b-CI to the CRIB domain of Ste20. These results suggest that the SH3b and the CI collaborate in tethering of Ste20 to Bem1 to ensure efficient mating pheromone signaling.
在酿酒酵母中，Rho 型小 GTP 酶 Cdc42 由其鸟嘌呤核苷酸交换因子 Cdc24 激活，使细胞极化以进行出芽和交配。多结构域蛋白 Bem1 不仅与 Cdc42 相互作用，还与 Cdc24 以及 Cdc42 的效应器相互作用，包括 p21 激活激酶 Ste20，作为细胞极性建立的支架发挥作用。尽管 Bem1 分别通过其 PB1 和第二个 SH3 结构域（SH3b）与 Cdc24 和 Ste20 相互作用，但尚不清楚 Bem1 如何结合 Cdc42。在这里，我们表明包含 SH3b 及其 C 端侧翼片段（称为 CI，即 SH3b-CI）的区域直接与 Cdc42 相互作用。双诱饵反向双杂交方法表明 CI 对这种相互作用至关重要：CI 中的 N253D 取代消除了 SH3b-CI 与 Cdc42 的结合，但不影响其与 Ste20 富含脯氨酸区域的结合，而 SH3b 中的 W192K 取代则产生相反的效果。然而，SH3b-CI 以互斥的方式与 Ste20 富含脯氨酸区域和 Cdc42 相互作用。N253D 取代使细胞生长对温度敏感并抑制交配。W192K 诱导的交配缺陷因 N253D 取代而加剧，并在 CI 完整的情况下通过增加 Ste20 的剂量而受到抑制。有趣的是，Cdc42 可以介导 SH3b-CI 与 Ste20 的CRIB 结构域的间接相互作用。这些结果表明，SH3b 和 CI 协同作用将 Ste20 拴系到 Bem1 上，以确保有效的交配信息素信号传导。
