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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.
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PMID:Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains. 1769 85

Interleukin 1 (IL-1) has been reported to stimulate the polyubiquitination and disappearance of IL-1 receptor-associated kinase 1 (IRAK1) within minutes. It has been thought that the polyubiquitin chains attached to IRAK1 are linked via Lys48 of ubiquitin, leading to its destruction by the proteasome and explaining the rapid IL-1-induced disappearance of IRAK1. In this paper, we demonstrate that IL-1 stimulates the formation of K63-pUb-IRAK1 and not K48-pUb-IRAK1 and that the IL-1-induced disappearance of IRAK1 is not blocked by inhibition of the proteasome. We also show that IL-1 triggers the interaction of K63-pUb-IRAK1 with NEMO, a regulatory subunit of the IkappaBalpha kinase (IKK) complex, but not with the NEMO[D311N] mutant that cannot bind K63-pUb chains. Moreover, unlike wild-type NEMO, the NEMO[D311N] mutant was unable to restore IL-1-stimulated NF-kappaB-dependent gene transcription to NEMO-deficient cells. Our data suggest a model in which the recruitment of the NEMO-IKK complex to K63-pUb-IRAK1 and the recruitment of the TAK1 complex to TRAF6 facilitate the TAK1-catalyzed activation of IKK by the TRAF6-IRAK1 complex.
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PMID:Interleukin-1 (IL-1) induces the Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitate NEMO binding and the activation of IkappaBalpha kinase. 1818 Feb 83

NF-kappaB transcription factors induce pro-inflammatory molecules (e.g. IL-8) in response to cytokines (e.g. TNFalpha, IL-1beta) or other stimuli. In the basal state, they are sequestered in the cytoplasm by inhibitory IkappaB proteins. Pro-inflammatory signaling triggers polyubiquitination of intermediaries (e.g. RIP1), which activate IkappaB kinases that trigger Ser phosphorylation and degradation of IkappaBalpha, thereby promoting nuclear translocation of NF-kappaB. A negative feedback loop exists whereby NF-kappaB drives resynthesis of IkappaBalpha, which promotes export of NF-kappaB from the nucleus to the cytoplasm. This process relies on Cezanne, a deubiquitinating cysteine protease that stabilizes resynthesized IkappaBalpha by removing polyubiquitin from modified intermediaries. H(2)O(2) is generated during inflammation. Here we examined the effects of H(2)O(2) on NF-kappaB dynamics and pro-inflammatory activation in cultured cells co-stimulated with TNFalpha or IL-1beta. Quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay revealed that H(2)O(2) enhanced the induction of IL-8 by TNFalpha or IL-1beta. We demonstrated by using assays of NF-kappaB nuclear localization and by imaging of live cells expressing a fluorescent form of NF-kappaB that H(2)O(2) prolonged NF-kappaB nuclear localization in cells co-stimulated with TNFalpha or IL-1beta by suppressing its export from the nucleus. We provide evidence that H(2)O(2) suppresses NF-kappaB export by prolonging polyubiquitination of signaling intermediaries, which promotes Ser phosphorylation and destabilization of newly synthesized IkappaBalpha proteins. Finally, we observed that the catalytic activity of Cezanne and its ability to suppress RIP1 polyubiquitination and NF-kappaB transcriptional activity were inhibited by H(2)O(2). We conclude that H(2)O(2) prolongs NF-kappaB activation in co-stimulated cells by suppressing the negative regulatory functions of Cezanne and IkappaBalpha.
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PMID:Hydrogen peroxide prolongs nuclear localization of NF-kappaB in activated cells by suppressing negative regulatory mechanisms. 1847 97

Attachment of ubiquitin to proteins represents a central mechanism for the regulation of protein metabolism and function. In the NF-kappaB pathway, binding of NEMO to polyubiquitinated substrates initiates the pathway in response to cellular stimuli. Other polyubiquitin binding proteins can antagonize this pathway by competing with NEMO for polyubiquitin. We have used protein arrays to identify polyubiquitin binding proteins that regulate NF-kappaB activity. Using polyubiquitin as bait, protein arrays were screened and polyubiquitin binders identified. Novel polyubiquitin binders AWP1, CALCOCO2, N4BP1, RIO3, TEX27, TTC3, UBFD1 and ZNF313 were identified using this approach, while known NF-kappaB regulators including NEMO, A20, ABIN-1, ABIN-2, optineurin and p62 were also identified. Overexpressed AWP1 and RIO3 repressed NF-kappaB activity in a manner similar to optineurin, while siRNAs directed against AWP1 and RIO3 also reduced NF-kappaB activity. TNFalpha-dependent degradation of IkappaBalpha was also suppressed by overexpression of AWP1 and RIO3, possibly due to the polyubiquitin binding activity of these proteins. Protein array screening using polyubiquitin enabled rapid identification of many known and novel polyubiquitin binding proteins and the identification of novel NF-kappaB regulators.
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PMID:Identification of polyubiquitin binding proteins involved in NF-kappaB signaling using protein arrays. 1928 59

IkappaBalpha serves as a central anchoring molecule in the sequestration of NF-kappaB transcription factor in the cytoplasm. Ubiquitination-mediated IkappaBalpha degradation immediately precedes and is required for NF-kappaB nuclear translocation and activation. However, the precise mechanism for the deubiquitination of IkappaBalpha is still not fully understood. Using a proteomic approach, we have identified Ubiquitin Specific Peptidase 11 (USP11) as an IkappaBalpha associated deubiquitinase. Overexpression of USP11 inhibits IkappaBalpha ubiquitination. Recombinant USP11 catalyzes deubiquitination of IkappaBalpha in vitro. Moreover, knockdown of USP11 expression enhances TNFalpha-induced IkappaBalpha ubiquitination and NF-kappaB activation. These data demonstrate that USP11 plays an important role in the downregulation of TNFalpha-mediated NF-kappaB activation through modulating IkappaBalpha stability. In addition, overexpression of a catalytically inactive USP11 mutant partially inhibits TNFalpha- and IKKbeta-induced NF-kappaB activation, suggesting that USP11 also exerts a non-catalytic function in its negative regulation of TNFalpha-mediated NF-kappaB activation. Thus, IkappaBalpha ubiquitination and deubiquitination processes function as a Yin-Yang regulatory mechanism on TNFalpha-induced NF-kappaB activation.
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PMID:USP11 negatively regulates TNFalpha-induced NF-kappaB activation by targeting on IkappaBalpha. 1987 89

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1.Cullin 1.F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206-215 and 216-225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys(48) and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe(206), Tyr(207), Tyr(210), and Tyr(211)) are probably positioned in the vicinity of ubiquitin C-terminal residue Val(70). Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val(70) by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1-215) but not Cdc34(1-200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IkappaBalpha ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe(206)/Tyr(207), Tyr(210)/Tyr(211)) in conjugation, with Tyr(210) exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr(210) was required for the transfer of the donor ubiquitin to a receptor lysine on either IkappaBalpha or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.
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PMID:The human Cdc34 carboxyl terminus contains a non-covalent ubiquitin binding activity that contributes to SCF-dependent ubiquitination. 2035 40


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