Modification by Single Ubiquitin Moieties Rather Than Polyubiquitination Is Sufficient for Proteasomal Processing of the p105 NF-κB Precursor

Abstract

Activation of NF-kappaB is regulated via numerous ubiquitin- and proteasome-mediated steps; an important one is processing of the precursor p105 to the p50 active subunit. The mechanisms involved are largely unknown, because this is an exceptional case where the ubiquitin system does not destroy its substrate completely. Here, we demonstrate that proteasomal processing of p105 requires ubiquitin but not generation of polyubiquitin chains. In vitro, ubiquitin species that cannot polymerize mediate processing. In yeasts that express nonpolymerizable ubiquitins, processing proceeds normally, whereas degradation of substrates that are dependent on polyubiquitination is inhibited. Similar results were obtained in mammalian cells. Interestingly, processing requires multiple monoubiquitinations, because progressive elimination of lysines in p105 is accompanied by gradual inhibition of p50 generation. Finally, the proteasome recognizes the multiply monoubiquitinated p105. These findings suggest that a proteolytic signal can be composed of a cluster of single ubiquitins, not necessarily a chain.

Ubiquitin degradation with its substrate, or as a monomer in a ubiquitination-independent mode, provides clues to proteasome regulation

“…The tail probably must be unstructured and f lexible to allow its entry and passage through the 19S complex, reaching the 20S complex (26). The lack of requirement for generation of a polyubiquitin chain in this and other cases (16,17) raises questions about the role of the chain that appears to be necessary for the targeting of most substrates. It is possible that the chain increases the affinity, and thus the efficiency of degradation of large substrates.…”

“…In vitro conjugation assays were carried essentially as described (13,16). Briefly, in vitrotranslated proteins (Ϸ30,000 cpm) were incubated at 37°C for 1 h (in a volume of 12.5 L) in the presence of HeLa cell extract (50 g), Ub (5 g), E1 (0.25 g), ATP␥S (0.5 mM), and the isopeptidase inhibitor, Ub aldehyde (100 ng).…”

“…Further studies on the mechanism of degradation of tailed Ub showed that it does not require further ubiquitination, highlighting in an as yet additional case the ability of the proteasome to recognize and degrade monoubiquitinated substrates (16,17). This was true also for UBB ϩ1 that was further extended to have a tail Ͼ19 residues.…”

“…This intermediate rebinds to the proteasome via its Ub moiety and via the tail reaches the catalytic chamber of the 20S complex, pulling the entire molecule behind it: Ub itself or Ub with a tail that is shorter than a certain size cannot reach the 20S-buried proteolytic sites. It can also represent the entire conjugated substrate that is degraded after monoubiquitination (16,17). An extended Ub can also represent naturally occurring derivatives.…”

Protein monoubiquitylation: targets and diverse functions

“…Second, p105 is monoubiquitylated at multiple lysine residues by CRL1 β‐TrCP , which is necessary for its processing to active p50 by the proteasome (Kravtsova‐Ivantsiv et al . 2009). Third, monoubiquitylation of NEMO at K277 and K309 by the E3 ligase cIAP1 in response to genotoxic stress triggers its export from the nucleus and assembly into the IKK complex, resulting in NF‐κB activation (Jin et al .…”