Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes.
The transcription factor c-Fos is a short-lived cellular protein. The levels of the protein fluctuate significantly and abruptly during changing pathophysiological conditions. Thus, it is clear that degradation of the protein plays an important role in its tightly regulated activity. We examined the involvement of the ubiquitin pathway in c-Fos breakdown. Using a mutant cell line, ts20, that harbors a thermolabile ubiquitin-activating enzyme, E1, we demonstrate that impaired function of the ubiquitin system stabilizes c-Fos in vivo. In vitro, we reconstituted a cell-free system and demonstrated that the protein is multiply ubiquitinated. The adducts serve as essential intermediates for degradation by the 26S proteasome. We show that both conjugation and degradation are significantly stimulated by c-Jun, with which c-Fos forms the active heterodimeric transcriptional activator AP-1. Analysis of the enzymatic cascade involved in the conjugation process reveals that the ubiquitin-carrier protein E2-F1 and its human homolog UbcH5, which target the tumor suppressor p53 for degradation, are also involved in c-Fos recognition. The E2 enzyme acts along with a novel species of ubiquitin-protein ligase, E3. This enzyme is distinct from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. We have purified the novel enzyme approximately 350-fold and demonstrated that it is a homodimer with an apparent molecular mass of approximately 280 kDa. It contains a sulfhydryl group that is essential for its activity, presumably for anchoring activated ubiquitin as an intermediate thioester prior to its transfer to the substrate. Taken together, our in vivo and in vitro studies strongly suggest that c-Fos is degraded in the cell by the ubiquitin-proteasome proteolytic pathway in a process that requires a novel recognition enzyme.
Department of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
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Stancovski
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Gonen
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Orian
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Schwartz
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Ciechanover
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D003546:Cysteine Endopeptidases / Q000378:metabolism; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D016762:Genes, fos; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008025:Ligases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis*; D027383:Papillomaviridae / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
C068651:E6 protein, Human papillomavirus type 16; D007457:Iodine Radioisotopes; C000715470:MAS1 protein, human; D009097:Multienzyme Complexes; D009856:Oncogene Proteins, Viral; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos; D011994:Recombinant Proteins; D012097:Repressor Proteins; D018808:Transcription Factor AP-1; D016159:Tumor Suppressor Protein p53; D017886:Durapatite; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes