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A proteomic screen reveals SCFGrr1 targets that regulate the glycolytic–gluconeogenic switch

Nature Cell Biology volume 9, pages 1184–1191 (2007)Cite this article

Abstract

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 (SCFGrr1) 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 SCFGrr1 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 SCFGrr1 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 SCFGrr1 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 SCFGrr1 in regulating the glycolytic–gluconeogenic switch.

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Figure 1: Identification of candidate Grr1 targets.
Figure 2: Regulation of Tye7, Stp1/2 and Pfk26/27.
Figure 3: Pfk27 is phosphorylated by Snf1.
Figure 4: Pfk27 turnover is important for the glycolytic–gluconeogenic switch.

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Acknowledgements

The authors thank J. Weissman, E. O'Shea, K. Weis, M. Tyers, A. Amon, C. Boone and T. Kishi for reagents; V. Vincent (Cellomics, Pittsburg, PA) and the University of California, San Francisco Laboratory for Cell Analysis for assistance with microscopy; members of the Morgan laboratory for reagents and discussion; and D. Morgan, K. Ashrafi, T. Fazzio and members of the Toczyski laboratory for discussion and comments on the manuscript. J.A.B is supported by the Damon Runyon Cancer Research Foundation. This work was supported by a National Institutes of Health grant to D.P.T.

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  1. Department of Biochemistry and Biophysics, Cancer Research Institute, University of California, San Francisco, 2340 Sutter Street, San Francisco, 94115, CA, USA

    Jennifer A. Benanti, Stephanie K. Cheung, Mariska C. Brady & David P. Toczyski

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  1. Jennifer A. Benanti
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  2. Stephanie K. Cheung
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Contributions

J.A.B. and D.P.T. designed the experiments, analysed the data and wrote the paper. J.A.B. carried out the experiments with assistance from S.K.C. and M.C.B.

Corresponding author

Correspondence to David P. Toczyski.

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Supplementary Information.

Supplementary Figures S1, S2, S3, S4 and S5, Supplementary Tables S1 and S2, and Methods (PDF 1048 kb)

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Benanti, J., Cheung, S., Brady, M. et al. A proteomic screen reveals SCFGrr1 targets that regulate the glycolytic–gluconeogenic switch. Nat Cell Biol 9, 1184–1191 (2007). https://doi.org/10.1038/ncb1639

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