Finding NEMO by K63-linked polyubiquitin chain

SCF-βTrCP-dependent K48-linked polyubiquitination has also been implicated in the generation of NF-κB p50 and p52 subunits from their respective precursors, p105 and p100. Both p105 and p100 proteins contain C-terminal ankyrin repeats similar to those in IκB proteins. Partial degradation of the C-terminal ankyrin repeats by proteasome results in the generation of mature forms of p50 and p52. The processing of p105 is dependent on IKKβ-mediated phosphorylation of S927 and S932, whereas the partial degradation of p100 requires phosphorylation of S866 and S870 by IKKα and its upstream kinase, NIK. Similarly, the processes of ubiquitination-dependent proteasomal degradation may be important in the elevation of NF-κB transcriptional activity in the nuclei.³ In this case, the protein targeted for ubiquitination and degradation is N-CoR, a ubiquitous nuclear receptor co-repressor, rather than IκB or NF-κB family proteins. N-CoR can associate with a number of transcription factors including NF-κB to repress their transcriptional activity. The biological consequences of N-CoR ubiquitination include the dismissal and subsequent degradation of N-CoR, which will lead to recruitment of the co-activators and transcriptional activation of target genes regulated by NF-κB or other transcription factors. The ubiquitin ligase responsible for N-CoR ubiquitination appears to be Siah, a RING-type family E3 ligase.⁴

Although the NF-κB field was abuzz with the activating role of protein ubiquitination in the earlier years, the opposite effect of ubiquitination on NF-κB was outlandish until the finding that p65 or c-Rel undergoes proteasomal degradation under certain circumstances. The first evidence indicating ubiquitination of NF-κB subunits is from a study of HTLV-1-infected T cells where ubiquitinated c-Rel proteins were noted.⁵ The C-terminal region of c-Rel was believed to be responsible for the ubiquitination and subsequent proteasome-mediated degradation of c-Rel. It remained obscure how such ubiquitination and degradation of c-Rel affects the activity of NF-κB. Following the observation of c-Rel ubiquitination, ubiquitin-dependent degradation of p65 was discovered in cells with deficiency in Pin1, a peptidyl-prolyl isomerase that isomerizes phosphorylated serine (S) or threonine (T) residues in S/T-P (proline) motif.⁶ In wild-type cells where the Pin1 is normally expressed, Pin1 is able to bind to the T254-phosphorylated p65 and stabilize p65 by preventing its ubiquitination and proteasomal degradation. Protein pull-down assay indicates an interaction between p65 and the suppressor of cytokine signaling 1 (SOCS-1).⁶ Based on the capability of SOCS-1 to interact with elongin B/C protein, SOCS-1 has been previously considered to be a receptor subunits (analogous to VHL) of VCB-like RING ubiquitin ligase complex. The association of SOCS-1 with COMMD1/Murr1, a protein involved in copper metabolism and NF-κB inhibition, has been implicated in the ubiquitination and degradation of p65, RelB, p50 and p52 in nuclei (Maine GN et al., personal communication). However, whether SOCS-1 is truly involved in the ubiquitination of p65 is under question, as this protein is mainly cytoplasmic.⁷ Given the fact that p65 contains a less conserved SCF-βTrCP docking site (536S-I-A-D-M540) and that S536 can be phosphorylated by either IKKβ or IKKα, the SCF-βTrCP may not be exonerated completely in the ubiquitination of p65. In addition, the STAT-interacting LIM protein (SLIM), a potential RING-type ubiquitin E3 ligase, may also play a role on the ubiquitination of p65 (T Tanaka and T Kaisho, personal communication). Ubiquitination of p65 is perhaps one of several mechanisms involved in the termination of activated NF-κB signals in inflammation resolution.⁸ It may also contribute to the observed oscillation of nuclear NF-κB activity or the exchange of NF-κB dimers following cytokine stimulation.⁹

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