Cytomegalovirus promoter up-regulation is the major cause of increased protein levels of unstable reporter proteins after treatment of living cells with proteasome inhibitors.
Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.
Alvarez-Castelao
Beatriz
B
Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols," Universidad Autónoma de Madrid y Consejo Superior de Investigaciones Científicas (UAM-CSIC) y Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina UAM, 28029 Madrid, Spain.
Martín-Guerrero
Idoia
I
Departamento de Genética, Antropología Física y Fisiología Animal, Facultad de Medicina, Universidad del País Vasco (UPV/EHU), 48940 Leioa, Spain.
García-Orad
África
Á
Departamento de Genética, Antropología Física y Fisiología Animal, Facultad de Medicina, Universidad del País Vasco (UPV/EHU), 48940 Leioa, Spain.
Castaño
José G
JG
Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols," Universidad Autónoma de Madrid y Consejo Superior de Investigaciones Científicas (UAM-CSIC) y Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina UAM, 28029 Madrid, Spain. Electronic address: joseg.castano@uam.es.
D000818:Animals; D002460:Cell Line; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D014176:Protein Biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation
D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins; D046988:Proteasome Endopeptidase Complex
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