Identification of Constitutive and Ras-Inducible Phosphorylation Sites of KSR: Implications for 14-3-3 Binding, Mitogen-Activated Protein Kinase Binding, and KSR Overexpression
Identification of Constitutive and Ras-Inducible Phosphorylation Sites of KSR: Implications for 14-3-3 Binding, Mitogen-Activated Protein Kinase Binding, and KSR Overexpression
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KSR 组成型和 Ras 诱导型磷酸化位点的鉴定：对 14-3-3 结合、丝裂原激活蛋白激酶结合和 KSR 过表达的影响
DOI：
10.1128/mcb.19.1.229
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发表时间：
1999
影响因子：
5.3
通讯作者：
D. Morrison
中科院分区：
文献类型：
--
作者：
A. Cacace;N. Michaud;M. Therrien;K. Mathes;T. Copeland;G. Rubin;D. Morrison
摘要Ras激酶抑制因子（KSR）是Ras依赖性信号通路中的一个保守组分。为了更好地理解KSR在信号转导中的作用，我们已经开始研究磷酸化和蛋白质相互作用对KSR功能的影响。在这里，我们报告的五个在体内KSR磷酸化位点的鉴定。在血清饥饿的细胞中，KSR含有两个组成性磷酸化位点（Ser 297和Ser 392），其介导KSR与14-3-3蛋白家族的结合。在激活的Ras存在下，KSR含有三个额外的磷酸化位点（Thr 260，Thr 274和Ser 443），所有这些位点都与丝裂原活化蛋白激酶（MAPK）磷酸化的共有基序（Px[S/T]P）相匹配。此外，我们发现用MEK抑制剂PD 98059处理细胞阻断Ras诱导位点的磷酸化，并且活化的MAPK以Ras依赖的方式与KSR相关联。总之，这些发现表明KSR是MAPK的体内底物。突变的磷酸化位点并没有改变KSR的能力，以促进Ras信号在非洲爪蟾卵母细胞，这表明在这些网站的磷酸化可能服务于其他功能的作用，如调节催化活性。有趣的是，在本研究过程中，我们发现KSR的生物学效应随着KSR蛋白表达水平的变化而变化。在非洲爪蟾卵母细胞中，KSR在低水平表达时作为Ras信号传导的正调节剂发挥作用，而在高水平表达时，KSR阻断Ras依赖的信号传导。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物的生物学功能似乎依赖于将KSR蛋白表达维持在低或接近生理水平。
ABSTRACT 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. InXenopus 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.