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.
Yen
Hsueh-Chi Sherry
HC
Department of Genetics, Center for Genetics and Genomics, Brigham and Women's Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.
Elledge
Stephen J
SJ
D017209:Apoptosis; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D055550:Protein Stability; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D020687:cdc25 Phosphatases / Q000302:isolation & purification / Q000378:metabolism; D000097573:Red Fluorescent Protein
D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D044843:SKP Cullin F-Box Protein Ligases; D020687:cdc25 Phosphatases; D000097573:Red Fluorescent Protein; C100318:Cullin 1; C485184:enhanced green fluorescent protein; C517004:CDC25A protein, human