title: The dark side of EGFP: defective polyubiquitination.
abstract: Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-kappaB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered.
authors.affiliation: Applied Human Genomics, Center for Human Genetics, Molecular Genetics-Flanders Interuniversity Institute for Biotechnology (VIB), University of Leuven, Belgium. Mathijs.Baens@Med.KULeuven.be
authors.forename: Mathijs
authors.initials: M
authors.lastname: Baens
authors.forename: Heidi
authors.initials: H
authors.lastname: Noels
authors.forename: Vicky
authors.initials: V
authors.lastname: Broeckx
authors.forename: Sofie
authors.initials: S
authors.lastname: Hagens
authors.forename: Sabine
authors.initials: S
authors.lastname: Fevery
authors.forename: An D
authors.initials: AD
authors.lastname: Billiau
authors.forename: Hugo
authors.initials: H
authors.lastname: Vankelecom
authors.forename: Peter
authors.initials: P
authors.lastname: Marynen
mesh_terms: D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008822:Mice, Transgenic; D016328:NF-kappa B / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D054875:Ubiquitination
chemical_list: C415943:API2-MALT1 fusion protein, human; D016328:NF-kappa B; D015514:Oncogene Proteins, Fusion; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D008239:Lysine