Cacace A M, Michaud N R, Therrien M, Mathes K, Copeland T, Rubin G M, Morrison D K
Molecular Basis of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Mol Cell Biol. 1999 Jan;19(1):229-40. doi: 10.1128/MCB.19.1.229.
Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.
遗传学和生物化学研究已确定Ras激酶抑制因子（KSR）是Ras依赖性信号通路的一个保守组成部分。为了更好地理解KSR在信号转导中的作用，我们启动了研究，以探讨磷酸化和蛋白质相互作用对KSR功能的影响。在此，我们报告了KSR的五个体内磷酸化位点的鉴定结果。在血清饥饿的细胞中，KSR含有两个组成型磷酸化位点（Ser297和Ser392），它们介导KSR与14-3-3蛋白家族的结合。在激活的Ras存在的情况下，KSR含有另外三个磷酸化位点（Thr260、Thr274和Ser443），所有这些位点都符合丝裂原活化蛋白激酶（MAPK）磷酸化的共有基序（Px[S/T]P）。此外，我们发现用MEK抑制剂PD98059处理细胞可阻断Ras诱导位点的磷酸化，并且活化的MAPK以Ras依赖性方式与KSR结合。这些发现共同表明KSR是MAPK的体内底物。所鉴定的磷酸化位点的突变并未改变KSR在非洲爪蟾卵母细胞中促进Ras信号传导的能力，这表明这些位点的磷酸化可能发挥其他功能作用，例如调节催化活性。有趣的是，在本研究过程中，我们发现KSR的生物学效应随所表达的KSR蛋白水平而显著变化。在非洲爪蟾卵母细胞中，当低水平表达时，KSR作为Ras信号传导的正调节因子发挥作用，而在高水平表达时，KSR阻断Ras依赖性信号转导。同样，果蝇KSR的过表达阻断了果蝇眼中R7光感受器的形成。因此，KSR作为Ras依赖性信号传导的正效应器的生物学功能似乎依赖于将KSR蛋白表达维持在低水平或接近生理水平。
