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-κB 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.
Highlights
The extensive use of Enhanced Green Fluorescent Protein (EGFP) as a live cell reporter is based on the presumption that it does not affect cellular functions
Constitutive activation of the transcription factor NF-kB is considered essential for B-cell transformation by the API2MALT1 fusion protein of MALT lymphomas with a translocation t(11;18) [10]
To our surprise we observed that EGFP inhibited NF-kB activation induced by API2-MALT1 in a dose dependent manner (Fig 1A)
Summary
The extensive use of EGFP as a live cell reporter is based on the presumption that it does not affect (important) cellular functions. EGFP did not affect reporter activation induced by expression of the p50/p65 subunits of NF-kB (Fig 1B), suggesting an interference with the NF-kB signalling cascade activated by API2-MALT1 upstream of NF-kB. Oligomerization of TRAF6 elicits the E3 ubiquitin ligase activity of its RING domain, resulting in modification of IKKc (NEMO) via Lys63-linked polyubiquitin chains.
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