Degradation of the Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) by the Ubiquitin-Proteasome Pathway

Abstract

The latent membrane protein 1 (LMP1) of the EpsteinBarr virus is a constitutively active receptor essential for B lymphocyte transformation by the Epstein-Barr virus. It is a short-lived protein, but the proteolytic pathway involved in its degradation is not known. The ubiquitin pathway is a major system for specific protein degradation in eukaryotes. Most plasma membrane substrates of the pathway are internalized upon ubiquitination and delivered for degradation in the lysosome/ vacuole. Here we show that LMP1 is a substrate of the ubiquitin pathway and is ubiquitinated both in vitro and in vivo. However, in contrast to other plasma membrane substrates of the ubiquitin system, it is degraded mostly by the proteasome and not by lysosomes. Degradation is independent of the single Lys residue of the protein; a lysine-less mutant LMP1 is degraded in a ubiquitin-and proteasome-dependent manner similar to the wild type protein. Degradation of both wild type and lysine-less protein is sensitive to fusion of a Myc tag to the N terminus of LMP1. In addition, deletion of as few as 12 N-terminal amino acid residues stabilizes the protein. These findings suggest that the first event in LMP1 degradation is attachment of ubiquitin to the N-terminal residue of the protein. We present evidence suggesting that phosphorylation is also required for degradation of LMP1.

Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue

“…Also, in cells, degradation of the lysine-less protein was sensitive to inhibition of the proteasome ( Figure 4). As no lysine residues were available for ubiquitination, we suspected that the ®rst ubiquitin residue is attached to the free N-terminal NH 2 group, as is the case for MyoD (Breitschopf et al, 1998) and EBV LMP1 (Aviel et al, 2000). To study the possibility that E7 is also targeted via initial N-terminal ubiquitination, and with the assumption that the N-terminal domain of the protein determines the speci®city for this process, we altered the N terminal domain of both WT and the lysine-less E7 by fusing it with a Myc-tag.…”

“…Similarly, ubiquitinmediated degradation of the Latent Membrane Protein 1 (LMP1) of the Epstein-Barr Virus is not dependent on the single internal Lys residue of the molecule, and also requires initial fusion of ubiquitin to the Nterminal residue (Aviel et al, 2000). To study whether a similar mechanism is also involved in the degradation of E7, we replaced the two Lys residues in positions 60 and 97 with Arg.…”

“…In most cases, the ®rst ubiquitin molecule is transferred to an e-NH 2 group of an internal Lysine residue of the target protein. However, targeting of the myogenic transcription factor MyoD (Breitschopf et al, 1998) and of the Epstein Barr Virus (EBV) Latent Membrane Protein-1 (LMP-1; Aviel et al, 2000) involves initial ubiquitination of the N-terminal residue followed by synthesis of a poly-ubiquitin chain attached to an internal Lys residue of this N-terminally attached ubiquitin moiety. Thus, unlike many known substrates of the ubiquitin system, degradation of these proteins does not require any internal Lys residue.…”

Ubiquitination of the Epstein–Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site

“…According to the authors, these findings do not exclude that the target proteins conjugated by K63 chains are not ultimately degraded, but they indicate that degradation, even if it occurs, is not necessary for IKK activation. Although LMP1 was shown to be degraded by a proteasome-dependent manner (Aviel et al, 2000), TRAF3 does not appear to be degraded after LMP1-induced signaling (Brown et al, 2001). Further studies will help us to understand whether the ubiquitination of LMP1 and TRAF3 plays a role in an activation process that is important for signaling.…”

“…LMP1 is a powerful inducer of NF-kB-mediated transcription (Hammarskjold and Simurda, 1992) and engages signaling proteins of the tumor necrosis factor receptor-associated factor (TRAF) family (Mosialos et al, 1995). LMP1 is a short-lived protein with a reported half-life ranging from 1.5 to 7 h, depending on the cell type used (Baichwal and Sugden, 1987;Mann and Thorley-Lawson, 1987;Martin and Sugden, 1991), is ubiquitinated and degraded by the proteasome (Aviel et al, 2000).…”