The Dark Side of EGFP: Defective Polyubiquitination

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

A Novel TRAF6 Binding Site in MALT1 Defines Distinct Mechanisms of NF-{kappa}B Activation by API2{middle dot}MALT1 Fusions

The recurrent translocation t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue (MALT) lymphoma results in the expression of an API2.MALT1 fusion protein that constitutively activates NF-kappaB. The first baculovirus IAP repeat (BIR) domain of API2 and the C terminus of MALT1, which contains its caspase-like domain, are present in all reported fusion variants and interact with TRAF2 and TRAF6, respectively, suggesting their contribution to NF-kappaB signaling by API2.MALT1. Also, the involvement of BCL10 has been suggested via binding to BIR1 of API2 and via its interaction with the immunoglobulin domains of MALT1, present in half of the fusion variants. However, conflicting reports exist concerning their roles in API2.MALT1-induced NF-kappaB signaling. In this report, streptavidin pulldowns of biotinylated API2.MALT1 fusion variants showed that none of the fusion variants interacted with endogenous BCL10; its role in NF-kappaB signaling by API2.MALT1 was further questioned by RNA interference experiments. In contrast, TRAF6 was essential for NF-kappaB activation by all fusion variants, and we identified a novel TRAF6 binding site in the second immunoglobulin domain of MALT1, which enhanced NF-kappaB activation when present in the fusion protein. Furthermore, inclusion of both immunoglobulin domains in API2.MALT1 further enhanced NF-kappaB signaling via intramolecular TRAF6 activation. Finally, binding of TRAF2 to BIR1 contributed to NF-kappaB activation by API2.MALT1, although additional mechanisms involving BIR1-mediated raft association are also important. Taken together, these data reveal distinct mechanisms of NF-kappaB activation by the different API2.MALT1 fusion variants with an essential role for TRAF6.status: publishe

EGFP affects polyubiquitination-dependent processes <b><i>in vivo</i></b>.

<div><p>(<b>A</b>) Western blots of spleen and liver extracts from FVB wild-type (wt) and EGFP transgenic mice detected with antibodies against Ubiquitin, EGFP and actin (loading control).</p> <p>(<b>B</b>) EGFP reduces phosphorylation of IκB-α in anti-IgM/anti-CD40 stimulated B-lymphocytes purified from EGFP mice.</p> <p>Experiments performed in triplicate, a representative image of one experiment is shown.</p> <p>The average and standard deviations of ratios of IκB-α-P to actin relative to un-stimulated cells are given.</p> <p>(<b>C</b>) The deficit of B220⁺/CD40⁺ B-cells in the bone marrow of API2-MALT1 mice is restored in EGFP/API2-MALT1 double transgenic mice.</p> <p>Experiments performed in triplicate, the average and standard deviations are depicted. eMalt1: endogenous Malt1, *a-specific band (<b>D</b>) EGFP mice show increased p53 levels in the liver and have enhanced p53/p21 responses upon γ-irradiation induced DNA damage.</p> <p>The average and standard deviations of the ratios of p53/p21 to actin signals relative to that of sample 1 are given.</p> <p>(<b>E</b>) Recombinant EGFP (rEGFP) does not prevent polyubiquitination of a Biotin-Lysozyme substrate <i>in vitro</i>. (Ub)ⁿ: polyubiquitinated proteins.</p></div

EGFP blocks Lys63- and Lys48-linked polyubiquitination.

<div><p>(<b>A</b>) 293T cells transfected with the indicated constructs and treated for 4 hours with 20 ng/ml TNF-α (lane 5 and 6) were immunoblotted with anti-Flag (API2-MALT1), anti-HA (HA-Ub-K63) and anti-EGFP antibodies (left panel) or anti-IKKγ immunoprecipitates were immunoblotted with anti-Flag (IKKγ) or anti-HA (ubiquitin) (right panel).</p> <p>(<b>B</b>) EGFP affects K48-linked polyubiquitination.</p> <p>293T cells were transfected with a Ubiquitin construct with only Lys48 available for polymerization (HA-Ub-K48), treated for 4 hours with 20 ng/ml TNF-α or left untreated and cell lysates were immunoblotted with anti-HA (Ub-K48) and anti-EGFP antibodies.</p> <p>Fluorescence intensities (Excitation 485/Emission 520 nm) for EGFP and pmaxGFP were comparable (∼100 fold higher then background values), expression of pDs-Red was confirmed by Fluorescence microscopy.</p> <p>(<b>C</b>) EGFP stabilizes exogenous API2-Myc in 293T cells via reduction of its Lys48-linked auto-ubiquitination and proteasomal degradation.</p> <p>(<b>D</b>) Stable expression of EGFP in the merkel cell carcinoma cell line MCC14.2 reduces polyubiquitination and enhances endogenous p53 expression levels.</p> <p>The average ratio and standard deviation of p53 to actin signals are given (three independent experiments), (Ub)ⁿ : polyubiquitinated proteins.</p></div