Xing H, Kornfeld K, Muslin A J
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Curr Biol. 1997 May 1;7(5):294-300. doi: 10.1016/s0960-9822(06)00152-7.
KSR (kinase suppressor of Ras) is a recently identified putative protein kinase that positively mediates the Ras signaling pathway in the invertebrates Caenorhabditis elegans and Drosophila melanogaster. The function of vertebrate KSR is not well characterized biochemically or biologically.
We examined the physiological role of KSR in vertebrate signal transduction using Xenopus laevis oocytes. Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induced Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation was blocked by co-expression of dominant-negative Raf-1. We noted that KSR contains multiple potential binding sites for 14-3-3, and we used the yeast two-hybrid system and co-immunoprecipitation experiments to show that KSR can bind to 14-3-3. Furthermore, we demonstrated that KSR can form a complex with Raf kinase both in vitro and in cultured cells. Cell fractionation studies revealed that KSR formed a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only formed a complex with Raf-1 in the membrane fraction.
Our finding suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms.
KSR（Ras激酶抑制因子）是最近鉴定出的一种假定的蛋白激酶，它在无脊椎动物秀丽隐杆线虫和黑腹果蝇中正向介导Ras信号通路。脊椎动物KSR的功能在生化或生物学方面尚未得到充分表征。
我们利用非洲爪蟾卵母细胞研究了KSR在脊椎动物信号转导中的生理作用。KSR的过表达与细胞内二聚体蛋白14-3-3的过表达相结合，诱导了非洲爪蟾卵母细胞的减数分裂成熟和cdc2激酶激活；KSR和14-3-3对卵母细胞成熟的作用被显性负性Raf-1的共表达所阻断。我们注意到KSR含有多个与14-3-3潜在的结合位点，并且我们使用酵母双杂交系统和免疫共沉淀实验表明KSR可以与14-3-3结合。此外，我们证明KSR在体外和培养细胞中都能与Raf激酶形成复合物。细胞分级分离研究表明，KSR在细胞裂解物的膜和细胞质部分均与14-3-3形成复合物；然而，KSR仅在膜部分与Raf-1形成复合物。
我们的发现表明，KSR、14-3-3和Raf形成一个寡聚信号复合物，并且KSR在脊椎动物中正向调节Ras信号通路。
