Mechanisms of ubiquitin-mediated, limited processing of the NF-kappaB1 precursor protein p105.
In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-kappaB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441-454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446-454 may serve as a ligase recognition motif. Following IkappaB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918-934, recruits the SCF(beta-TrCP) ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-kappaB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IkappaBgamma). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.
Ciechanover
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Department of Biochemistry and the Rappaport Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Haifa, Israel. c_tzachy@netvision.net.il
Gonen
H
H
Bercovich
B
B
Cohen
S
S
Fajerman
I
I
Israël
A
A
Mercurio
F
F
Kahana
C
C
Schwartz
A L
AL
Iwai
K
K
Orian
A
A
D020816:Amino Acid Motifs; D017487:Ankyrins; D004268:DNA-Binding Proteins / Q000378:metabolism; D005998:Glycine; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010453:Peptide Synthases / Q000378:metabolism; D010766:Phosphorylation; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases; D015398:Signal Transduction / Q000502:physiology; D014452:Ubiquitins / Q000378:metabolism*
D017487:Ankyrins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; D011498:Protein Precursors; D014452:Ubiquitins; D044843:SKP Cullin F-Box Protein Ligases; C090571:ubiquitin carrier protein kinase; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D010447:Peptide Hydrolases; D010453:Peptide Synthases; D005998:Glycine