Altering Drosophila S6 kinase activity is consistent with a role for S6 kinase in growth
Altering Drosophila S6 kinase activity is consistent with a role for S6 kinase in growth
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DOI：
10.1002/gene.10132
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发表时间：
2002-09-01
期刊：
影响因子：
1.5
通讯作者：
Stewart, MJ
中科院分区：
文献类型：
--
作者：
Barcelo, H;Stewart, MJ
控制生长对于多细胞生物体的发育至关重要，并且器官或生物体的大小可以通过细胞的数量和/或单个细胞的大小来确定。最近的一些研究表明，调节生长的信号通路是高度保守的。在哺乳动物和果蝇中，胰岛素或胰岛素样信号通过PI 3-K信号通路调节细胞大小、器官大小和细胞增殖（约翰斯顿和Gallant，2002; Miron和Sonenberg，2001）。主要在哺乳动物细胞中进行的研究表明，通过PI 3-K途径和蛋白激酶TOR的信号传导通过促进p70核糖体蛋白S6激酶（S6 K1）和真核翻译起始因子4 E结合蛋白4 E-BP 1的调节来调节生长所需的蛋白质合成。4 E-BP 1的磷酸化对于帽依赖性蛋白质合成的起始是重要的（Gingras等人，1998），而S6 K1被认为在调节翻译机器的产生中具有关键作用（Dufner和托马斯，1999）。一旦激活，S6 K1磷酸化40 S核糖体亚基蛋白S6，这导致通常编码核糖体蛋白和翻译起始因子的5 TOP mRNA的选择性翻译。哺乳动物S6 K1和果蝇S6 K1同源物dS6 K的突变分析表明，这些激酶通过调节细胞大小而不是细胞数量来调节生长。大多数果蝇是一个假定的无效dS6 K等位基因，dS6 Kl-1纯合，在发育过程中死亡，但少数出现5天的发育延迟后的成年人。dS6 K突变果蝇的比例正常，但小于其杂合同胞或野生型果蝇（Montagne等，1999年）。对成年翅膀细胞的检查表明，dS6 Kl-1突变导致细胞大小减少30%，而不影响翅膀中细胞的数量。类似地，敲除S6 K1基因的纯合子小鼠发育延迟，但发育为正常比例的小小鼠。有趣的是，在这些小鼠中，S6磷酸化和5 TOP mRNA翻译不受影响，这一结果可以通过小鼠中第二种S6激酶S6 K2的补偿作用来解释（Shima et al.，1998年）。S6 K1的活性受多种丝氨酸（S）和苏氨酸（T）的磷酸化调节，其中许多丝氨酸和苏氨酸在dS6 K中是保守的（图1）（Stewart et al.，1996年）。S6 K1的激活被认为涉及一系列连续的磷酸化事件，其开始于激酶的自抑制结构域中S411、S418、T421和S424的磷酸化（Pullen和托马斯，1997）。四个自身抑制位点的磷酸化被认为使S6 K1稳定在允许随后在S6 K1连接结构域中的T389磷酸化的构象中。这又被认为诱导S6 K1的构象，其允许组成型活性PDK 1接近并磷酸化T229（Dennis等人，1996; Pullen和托马斯，1997）。TOR与S411、T421、S424、T389的磷酸化有关（Isotani等人，1999）和T371（Saitoh等人，2002年）。然而，TOR在调节S6 K1活性中的作用尚未解决，因为其他研究表明TOR可以通过负调节抑制S6 K1的磷酸酶来激活S6 K1（Hara等人，1998; Peterson等人，1999年）。为了更详细地研究果蝇S6激酶在信号传导和生长中的作用，我们已经产生了编码dS6 K变体的dS6 K cDNA，我们预测这些变体会改变活性（图1）。将这些cDNA克隆到载体pUAST中，并用于产生转基因果蝇，其中UAS-dS6 K转基因表达被抑制。
Controlling growth is critically important to the development of multicellular organisms and the size of an organ or an organism can be determined by the number of cells and/or by the size of individual cells. A number of recent studies have shown that the signaling pathways that regulate growth are highly conserved. In mammals and Drosophila, insulin-or insulin-like signaling regulates cell size, organ size, and cell proliferation via the PI3-K signaling pathway (Johnston and Gallant, 2002; Miron and Sonenberg, 2001). Studies done primarily in mammalian cells indicate that signaling through the PI3-K pathway and the protein kinase TOR regulate protein synthesis, which is required for growth, by contributing to the regulation of the p70 ribosomal protein S6 kinase (S6K1) and the eukaryotic translation initiation factor 4E binding protein, 4E-BP1. Phosphorylation of 4E-BP1 is important for the initiation of cap-dependent protein synthesis (Gingras et al., 1998) while S6K1 is thought to have a key role in regulating production of the translational machinery (Dufner and Thomas, 1999). Upon activation, S6K1 phosphorylates the 40S ribosomal subunit protein S6 and this leads to the selective translation of 5TOP mRNAs that generally encode ribosomal proteins and translation initiation factors. Mutational analysis of mammalian S6K1 and the Drosophila S6K1 homolog, dS6K, has shown that these kinases regulate growth by regulating cell size but not cell number. Most flies that are homozygous for a putative null dS6K allele, dS6Kl-1, die during development, but a few emerge as adults after a 5-day developmental delay. dS6K mutant flies are normally proportioned but smaller than their heterozygous siblings or wild-type flies (Montagne et al., 1999). Examining cells of adult wings showed that the dS6Kl-1 mutation causes a reduction in cell size by 30% without affecting the number of cells in the wing. Similarly, mice homozygous for a knockout of the S6K1 gene are developmentally delayed, but develop as normally proportioned, small mice. Interestingly, in these mice S6 phosphorylation and 5TOP mRNA translation are not affected, a result that may be explained by the compensatory action of a second S6 kinase in mice, S6K2 (Shima et al., 1998). The activity of S6K1 is regulated by the phosphorylation of multiple serines (S) and threonines (T), many of which are conserved in dS6K (Fig. 1)(Stewart et al., 1996). Activation of S6K1 is thought to involve a sequential series of phosphorylation events that begins with phosphorylation of S411, S418, T421, and S424 in the autoinhibitory domain of the kinase (Pullen and Thomas, 1997). Phosphorylation of the four autoinhibitory sites is thought to stabilize S6K1 in a conformation that allows the subsequent phosphorylation of T389 in the S6K1 linker domain. This, in turn, is thought to induce a conformation of S6K1 that allows the constitutively active PDK1 to access and phosphorylate T229 (Dennis et al., 1996; Pullen and Thomas, 1997). TOR has been implicated in phosphorylating S411, T421, S424, T389 (Isotani et al., 1999), and T371 (Saitoh et al., 2002). The role of TOR in regulating S6K1 activity, however, is unresolved, as other studies suggest that TOR may activate S6K1 by negatively regulating a phosphatase that inhibits S6K1 (Hara et al., 1998; Peterson et al., 1999). To create the potential for more detailed studies of Drosophila S6 kinase in signaling and growth, we have generated dS6K cDNAs that encode dS6K variants that we predicted would have altered activity (Fig. 1). These cDNAs were cloned into the vector pUAST and used to generate transgenic flies in which UAS-dS6K transgene expression is …