Benanti Jennifer A, Cheung Stephanie K, Brady Mariska C, Toczyski David P
Department of Biochemistry and Biophysics, Cancer Research Institute, University of California, San Francisco, 2340 Sutter Street, San Francisco, CA 94115, USA.
Nat Cell Biol. 2007 Oct;9(10):1184-91. doi: 10.1038/ncb1639. Epub 2007 Sep 9.
Entry into the cell cycle is regulated by nutrient availability such that cells do not divide when resources are limited. The Skp1-Cul1-F-box (SCF) ubiquitin ligase with the F-box protein Grr1 (SCF(Grr1)) controls the proteolytic turnover of regulators of cell-cycle entry and a glucose sensor, suggesting that it links the cell cycle with nutrient availability. Here, we show that SCF(Grr1) broadly regulates cellular metabolism. We have developed a proteomic screening method that uses high-throughput quantitative microscopy to comprehensively screen for ubiquitin-ligase substrates. Seven new metabolic targets of SCF(Grr1) were identified, including two regulators of glycolysis--the transcription factor Tye7 and Pfk27. The latter produces the second messenger fructose-2,6-bisphosphate that activates glycolysis and inhibits gluconeogenesis. We show that SCF(Grr1) targets Pfk27 and Tye7 in response to glucose removal. Moreover, Pfk27 is phosphorylated by the kinase Snf1, and unphosphorylatable Pfk27 is stable and inhibits growth in the absence of glucose. These results demonstrate a role for SCF(Grr1) in regulating the glycolytic-gluconeogenic switch.
细胞周期的进入受营养物质可用性的调节，因此当资源有限时细胞不会分裂。带有F盒蛋白Grr1的Skp1-Cul1-F盒（SCF）泛素连接酶（SCF(Grr1)）控制细胞周期进入调节因子和葡萄糖传感器的蛋白水解周转，这表明它将细胞周期与营养物质可用性联系起来。在这里，我们表明SCF(Grr1)广泛调节细胞代谢。我们开发了一种蛋白质组学筛选方法，该方法使用高通量定量显微镜全面筛选泛素连接酶底物。鉴定出了SCF(Grr1)的七个新的代谢靶点，包括糖酵解的两个调节因子——转录因子Tye7和Pfk27。后者产生激活糖酵解并抑制糖异生的第二信使果糖-2,6-二磷酸。我们表明SCF(Grr1)在葡萄糖去除时靶向Pfk27和Tye7。此外，Pfk27被激酶Snf1磷酸化，并且不可磷酸化的Pfk27是稳定的，并且在没有葡萄糖的情况下抑制生长。这些结果证明了SCF(Grr1)在调节糖酵解-糖异生转换中的作用。
