Identification of SCF Ubiquitin Ligase Substrates by Global Protein Stability Profiling

Ubiquitin-mediated proteolysis regulates all aspects of cellular function, and defects in this process are associated with human diseases. The limited number of identified ubiquitin ligase–substrate pairs is a major bottleneck in the ubiquitin field. We established and applied genetic technologies that combine global protein stability (GPS) profiling and genetic perturbation of E3 activity to screen for substrates of the Skp1–cullin–F-box (SCF) ubiquitin ligase in mammalian cells. Among the >350 potential substrates identified, we found most known SCF targets and many previously unknown substrates involved in cell cycle, apoptosis, and signaling pathways. Exploring cell cycle–stage stability, we found that several substrates used the SCF and other E3s in different cell cycle stages. Our results demonstrate the potential of these technologies as general platforms for the global discovery of E3-substrate regulatory networks.

[1]  Cuiling Li,et al.  Docking of Axonal Mitochondria by Syntaphilin Controls Their Mobility and Affects Short-Term Facilitation , 2008, Cell.

[2]  B. Bartel,et al.  FKF1, a Clock-Controlled Gene that Regulates the Transition to Flowering in Arabidopsis , 2000, Cell.

[3]  Timothy Cardozo,et al.  The SCF ubiquitin ligase: insights into a molecular machine , 2004, Nature Reviews Molecular Cell Biology.

[4]  S. Elledge,et al.  A family of mammalian F-box proteins , 1999, Current Biology.

[5]  Zhijian J. Chen,et al.  Signal-induced ubiquitination of IκBα by the F-box protein Slimb/β-TrCP , 1999 .

[6]  M. Whitfield,et al.  The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing. , 1996, Genes & development.

[7]  Brett Larsen,et al.  The cell-cycle regulatory protein Cks1 is required for SCFSkp2-mediated ubiquitinylation of p27 , 2001, Nature Cell Biology.

[8]  Jianping Jin,et al.  Identification of substrates for F-box proteins. , 2005, Methods in enzymology.

[9]  Mike Tyers,et al.  F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex , 1997, Cell.

[10]  J. M. Boyd,et al.  Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins , 1994, Cell.

[11]  David P. Toczyski,et al.  A proteomic screen reveals SCFGrr1 targets that regulate the glycolytic–gluconeogenic switch , 2007, Nature Cell Biology.

[12]  G. Struhl,et al.  Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb , 1998, Nature.

[13]  M. Kirschner,et al.  The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. , 2000, Genes & development.

[14]  V. Scheuss,et al.  Syntaphilin A Syntaxin-1 Clamp that Controls SNARE Assembly , 2000, Neuron.

[15]  Joseph S. Takahashi,et al.  Circadian Mutant Overtime Reveals F-box Protein FBXL3 Regulation of Cryptochrome and Period Gene Expression , 2007, Cell.

[16]  C. Borchers,et al.  Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase , 2003, Molecular and Cellular Biology.

[17]  M. Wolter,et al.  Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC , 2002, International journal of cancer.

[18]  Qikai Xu,et al.  Global Protein Stability Profiling in Mammalian Cells , 2008, Science.

[19]  S. Elledge,et al.  Phosphorylation-Dependent Ubiquitination of Cyclin E by the SCFFbw7 Ubiquitin Ligase , 2001, Science.

[20]  Lianfa Shi,et al.  Nix and Nip3 Form a Subfamily of Pro-apoptotic Mitochondrial Proteins* , 1999, The Journal of Biological Chemistry.

[21]  R. Bruick.  Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Peter,et al.  Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Daniel A. Haber,et al.  Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines , 2001, Nature.

[24]  L. Vassilev,et al.  In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 , 2004, Science.

[25]  Angelika Amon,et al.  The regulation of Cdc20 proteolysis reveals a role for the APC components Cdc23 and Cdc27 during S phase and early mitosis , 1998, Current Biology.

[26]  S. Elledge,et al.  Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex , 2002, Nature.

[27]  P. Howley,et al.  Ubiquitination and degradation of the substrate recognition subunits of SCF ubiquitin-protein ligases. , 1998, Molecular cell.

[28]  K. Nakayama,et al.  Ubiquitin ligases: cell-cycle control and cancer , 2006, Nature Reviews Cancer.

[29]  A Ciechanover,et al.  The ubiquitin-proteasome pathway and pathogenesis of human diseases. , 1999, Annual review of medicine.

[30]  Stephen J. Elledge,et al.  SKP1 Connects Cell Cycle Regulators to the Ubiquitin Proteolysis Machinery through a Novel Motif, the F-Box , 1996, Cell.

[31]  Stephen J. Elledge,et al.  The SCFβ-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro , 1999 .

[32]  M. Kirschner,et al.  Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. , 1998, Molecular cell.

[33]  Michele Pagano,et al.  SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27 , 1999, Nature Cell Biology.

[34]  G. Nalepa,et al.  Therapeutic anti-cancer targets upstream of the proteasome. , 2003, Cancer treatment reviews.

[35]  Takashi Takata,et al.  Role of Cks1 overexpression in oral squamous cell carcinomas: cooperation with Skp2 in promoting p27 degradation. , 2004, The American journal of pathology.

[36]  T. Akiyama,et al.  Inhibition of Wnt signaling by ICAT, a novel β-catenin-interacting protein , 2000, Genes & Development.

[37]  D. Thomas,et al.  A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif. , 1998, Molecular cell.