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The transcription factor NF-κB (nuclear factor kappa enhancer binding protein) controls many processes including immunity, inflammation and apoptosis. Studies of the NF-κB pathway have revealed new functions for ubiquitination in addition to its well-known role in targeting protein degradation. Ubiquitin is involved in at least three steps in this pathway: degradation of the NF-κB inhibitor IκB, processing of NF-κB precursors, and activation of the IκB kinase (IKK) through a degradation-independent mechanism.
In this post-genomic era filled with cutting-edge technologies, it is refreshing to look back at the history of ubiquitin and NF-κB as both were discovered through an ingenious combination of simple "bucket" biochemistry and a clear mind1–5. Since these classic experiments, both ubiquitin and NF-κB have taken center stage in biology, as each controls a vast array of cellular and organismal processes ranging from the birth and death of a cell to the neuronal behavior of an organism. Again, it was through biochemistry that the essential role of ubiquitination in NF-κB activation was revealed6–8. Ubiquitin targets not only IκB for degradation, but also the NF-κB precursors, p105 and p100, for processing into mature forms by the proteasome. More surprisingly, a critical role for ubiquitination in activating protein kinases in the NF-κB pathway through a degradation-independent mechanism has also been discovered9–11. Thus, the convergence of ubiquitin and NF-κB research has not only helped elucidate the biochemical mechanism underlying NF-κB activation, but also broadened our understanding of ubiquitin as a versatile signaling tag. As several extensive reviews of ubiquitination in the NF-κB pathway are available12,13, here I will summarize the current view of the traditional role of ubiquitin in the NF-κB pathway, and then focus on recent advances in understanding the non-traditional role of ubiquitin in this pathway.
NF-κB is a dimeric transcription factor consisting of Rel family members, which include Rel-A (also known as p65), c-Rel, Rel-B, p50 and p5214. p50 and p52 are derived from the larger precursors p105 and p100, respectively, through proteolytic processing by the proteasome. All NF-κB proteins contain a highly conserved Rel-homology domain (RHD) that is responsible for DNA binding, dimerization, nuclear translocation and interaction with the IκB proteins. The IκB proteins, including IκBα, β and , bind to NF-κB via ankyrin repeats and block its nuclear import and thereby, its transcriptional activity. The C-termini of p105 and p100 also contain the IκB-like ankyrin repeats that must be degraded in order to generate the mature Rel subunits.
The NF-κB activation pathways are broadly classified as the canonical and non-canonical pathways, depending on whether activation involves IκB degradation or p100 processing (Figure 1)15. In the canonical pathway, which is the predominant NF-κB signaling pathway, stimulating cells with an agonist such as tumor necrosis factor α (TNFα) or interleukin-1β (IL-1β) activates the IKK complex that is composed of two catalytic subunits IKKα and IKKβ and a regulatory subunit NEMO (also known as IKKγ). Genetic experiments have demonstrated that IKKβ, but not IKKα, phosphorylates IκB proteins at two N-terminal serine residues. This signal-induced phosphorylation targets IκB for polyubiquitination and subsequent degradation by the proteasome7, thus releasing NF-κB. The non-canonical pathway of NF-κB activation operates mainly in B cells in response to stimulation of a subset of the TNF receptor superfamily, including receptors for BAFF, lymphotoxin-β (LTβ) and CD40 ligand. Stimulation of these receptors activates the protein kinase NIK, which in turn activates IKKα. IKKα then phosphorylates p100 at two C-terminal serine residues leading to the selective degradation of its IκB -like domain by the proteasome16,17. The mature p52 subunit and its binding partner Rel-B translocate into the nucleus to regulate gene expression.
The ubiquitin-proteasome pathway plays a crucial role in both the canonical and non-canonical pathways of NF-κB activation. Ubiquitination is a reversible covalent modification catalyzed by three enzymatic steps18. In the first step, ubiquitin is activated by a ubiquitin-activating enzyme (E1) in an ATP-dependent reaction. In the second step, the activated ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2 or Ubc), forming an E2-Ub thioester. Finally, in the presence of a ubiquitin-protein ligase (E3), ubiquitin is attached to a target protein through an isopeptide bond between the carboxyl terminus of ubiquitin and the -amino group of a lysine residue in the target protein. Ubiquitin contains seven lysines, which can be attached to another ubiquitin in a highly processive reaction to form a polyubiquitin chain. Typically, a polyubiquitin chain that targets a protein for degradation by the proteasome is linked through lysine-48 (K48) of ubiquitin19. However, ubiquitin chains linked through other lysines of ubiquitin have also been found in cells20. In particular, K63-linked polyubiquitin chains have recently been found to regulate DNA repair and protein kinase activation through a degradation-independent mechanism (see below).
Ubiquitination of IκB is carried out by an E2 of the Ubc4/5 family7,9,21 and the SCF-βTrCP E3 ligase (Skp1-Cul1-F-box ligase containing the F-box protein βTrCP)22–26. Two βTrCP proteins, βTrCP1 and βTrCP2, have been found in mammalian cells. Genetic deletion of βTrCP1 in mice leads to only modest retardation of IκBα degradation, suggesting that βTrCP1 and βTrCP2 may have redundant functions27. Indeed, silencing of both βTrCP1 and βTrCP2 expression by RNAi block IκBα degradation. βTrCP1 and βTrCP2 bind specifically to the phosphorylated form of IκB through their C-terminal WD40 repeats, and to the rest of the SCF complex through the F-box. The SCF complex contains the RING domain protein Roc1/Rbx1, which binds to Ubc4/5, allowing this E2 to ubiquitinate IκB at two conserved N-terminal lysine residues. The polyubiquitinated IκB remains associated with NF-κB, but is selectively degraded by the 26S proteasome, while NF-κB itself is spared7.
The ubiquitin-proteasome pathway is also responsible for the processing of p105 and p100 to p50 and p52, respectively. p105 can be processed either co-translationally or post-translationally, both requiring the proteasome6,28. Co-translational processing of p105 appears to be a constitutive process that does not require phosphorylation or ubiquitination and is likely the major source of p50, which is constitutively present in unstimulated cells. Some agents such as phorbol ester (PMA) and lipopolysaccharides (LPS) can stimulate post-translational processing of p105 by activating IKK, which phosphorylates p105 at a C-terminal domain29. On the other hand, the processing of p100 is tightly regulated by the non-canonical pathway of NF-κB activation and depends on both phosphorylation and ubiquitination. As discussed above, stimulation of certain receptors of the TNFR family on B cells activates IKKα, which phosphorylates p100 at two C-terminal serine residues (Figure 1). This phosphorylation relieves the inhibition of p100 processing by a C-terminal death domain, and recruits the SCF-βTrCP ligase to polyubiquitinate p100 at a specific lysine 16,17,30,31. Subsequently, the C-terminal IκB-like domain is selectively degraded by the proteasome, generating the mature p52 subunit.
How does the proteasome partially degrade a protein? More specifically, what defines where degradation starts and stops? A plausible model for regulated ubiquitin/proteasome-dependent processing has recently been proposed 32. According to this model (Figure 2), the ubiquitin tag on a substrate recruits the proteasome to an internal unstructured sequence such as the glycine rich region (GRR) of p105 and p10033. It is postulated that this region forms a hairpin-like loop that inserts into the proteolytic chamber of the 20S proteasome. Proteolysis begins at the loop and then proceeds in both the N- and C-terminal directions of the target protein. In most cases, the N- and C-terminal domains can be completely unfolded by the 19S subcomplex of the proteasome and threaded into the proteolytic chamber so that the target is completely degraded. In the cases of substrates such as p105 and p100, the RHD domain is a tightly folded structure that may be difficult to unfold. Thus, after initiating proteolysis from the putative GRR loop of p105 or p100, the proteasome chews along the C-terminal polypeptide until it reaches the end, but comes to a halt at the N-terminus when it encounters the tightly folded RHD structure.
Serendipity played a major role in the discovery of a ubiquitination-dependent IκB kinase complex9. In 1995, a cell-free system was established to demonstrate the signal-induced ubiquitination of IκBα7. This system was later used to identify the enzymes involved in IκB ubiquitination by separating HeLa cytosolic extracts into two fractions, dubbed fraction I and II1,9. Surprisingly, each fraction alone no longer supported IκBα phosphorylation but when both were combined, or when fraction I was replaced with ubiquitin and Ubc4 or Ubc5, IκBα phosphorylation was restored. Further purification of fraction II led to the identification of a 700 kDa kinase complex whose activity can be regulated by polyubiquitination in a degradation-independent manner9.
A major issue concerning the early work on IKK activation by ubiquitin was the lack of a connection between ubiquitination and signaling pathways known to activate IKK. This issue was largely resolved by the discovery that TRAF (TNF receptor associated factor) proteins are ubiquitin E3 ligases10. TRAF proteins play a pivotal role in signaling pathways for NF-κB activation from many cell surface receptors including the TNF receptor superfamily (TNFR), IL-1 receptors (IL-1R) and Toll-like receptors (TLRs)34. Seven members of this family have been identified in the human genome. With the exception of TRAF1, all TRAF proteins contain an N-terminal RING domain followed by several zinc finger domains. Among all TRAF proteins, TRAF2 and 6 have been most intensively studied. TRAF2 is recruited to the TNF receptor complex through binding to the adaptor protein TRADD35. Both TRADD and TRAF2 also interact with the receptor interacting protein RIP, a protein kinase that signals in the TNF pathway through a kinase-independent mechanism. TRAF6 is essential for IKK and JNK activation in the IL-1 and TLR pathways36,37. In these pathways, stimulation of the receptors leads to the recruitment of MyD88, an adaptor protein that further recruits the protein kinase IRAK4 and IRAK1. IRAK1 then binds to TRAF6, which subsequently activates downstream signaling cascades including those of IKK and JNK.
In a series of biochemical experiments aiming at elucidating the mechanism of IKK activation by TRAF6, two TRAF6-regulated IKK activators (TRIKAs) were identified10,11. TRIKA1 is an E2 enzyme complex consisting of Ubc13 and a Ubc-like protein Uev1A10,38. This E2 functions with TRAF6, a RING domain E3, to synthesize a K63-linked polyubiquitin chain on target proteins including NEMO and TRAF6 itself. TRIKA2 is a trimeric complex consisting of the protein kinase TAK1 and two adaptor proteins TAB1 and TAB211,39. TAK1 phosphorylates IKKβ at two serine residues in the activation loop, thereby activating IKK. TAB2 and its related protein TAB3 contain a highly conserved novel zinc finger (NZF) domain that binds preferentially to K63-linked polyubiquitin chains40. Mutation of the NZF domain abolishes the ability of TAB2 and TAB3 to activate TAK1 and IKK, whereas the replacement of the NZF domain with a heterologous ubiquitin-binding domain restores the function of TAB2 and TAB3, suggesting that ubiquitin-binding by the NZF domain is necessary for activating TAK1 and IKK. These studies have led to the following model of IKK activation by TRAF6-mediated polyubiquitination (Figure 3). Upon ligand binding to IL-1R or TLRs, TRAF6 is recruited to the receptor complexes and forms oligomers. TRAF6 oligomerization activates its ligase activity, leading to K63-linked polyubiquitination of targets including TRAF6 itself. Ubiquitinated TRAF6 recruits TAB2 and activates the TAB2-associated TAK1 kinase, which then phosphorylates and activates IKK. Ubiquitin-activated TAK1 also phosphorylates and activates MKK kinases such as MKK6, which in turn activates the JNK and p38 kinase pathway11. Thus, TRAF6-mediated polyubiquitination initiates protein kinase cascades through a degradation-independent mechanism. Recent structural studies have shown that the K48- and K63-linked ubiquitin chains adopt very different configurations, potentially providing the molecular basis for the distinct signaling functions of these polyubiquitin chains41.
Like TRAF6, TRAF2 is also a RING domain ubiquitin ligase (Figure 4)40,42–44. A dominant negative mutant of Ubc13 has been shown to block NF-κB activation by TRAF2 or TNFα10. Overexpression of TRAF2 leads to its polyubiquitination by Ubc13/Uev1A, and TRAF2 ubiquitination appears to be important for the activation of JNK42. A recent study showed that TRAF2 ubiquitination is essential for JNK, but not p38 or NF-κB, activation by TNFα44. This is likely due to the redundant role of TRAF2 and TRAF5 in NF-κB activation by TNFα, as TRAF2-deficient cells are defective in JNK but not NF-κB activation45, whereas TRAF2/TRAF5 double knockout cells are defective in both JNK and NF-κB activation46. TRAF2 and/or TRAF5 may also ubiquitinate RIP, which is essential for NF-κB activation40,47. It has been shown that TNFα induces RIP polyubiquitination, and that ubiquitinated RIP associates with the TNF receptor as well as TAB240,48,49. Thus, it is possible that polyubiquitination of RIP recruits the TAK1/TAB2 complex, which subsequently activates IKK. However, definitive proof that RIP polyubiquitination is required for TNFα signaling is still lacking.
Since TAK1 knockout mice are early embryonic lethal, it has not been possible to determine whether this kinase is essential for NF-κB activation in mice50. However, multiple lines of evidence support a crucial role for the TAK1 complex in IKK and JNK activation. First, TAK1 phosphorylates IKKβ and MKK6 at key serine residues within the activation loop, leading to their activation 11. Second, silencing of TAK1 expression by RNAi or inhibition with a specific kinase inhibitor in mammalian cells drastically reduced IKK activation and completely blocked JNK activation by IL-1β and TNFα40,51,52. Third, mutations of TAK1 in Drosophila melanogaster, or silencing of TAK1 in a D. melanogaster cell line, blocked IKK and JNK in response to bacterial challenge53–55. Thus, independent genetic and biochemical studies have indicated that TAK1 is important for IKK and JNK activation in both invertebrates and vertebrates. With regard to the role of TAB2 in the NF-κB pathway, a recent study showed that a TAB2-deficient cell line has normal NF-κB activation in response to IL-1β56. However, this is likely due to the redundant functions of TAB2 and its closely related homologue TAB3, as RNAi against TAB2 and TAB3 in mammalian cells abrogated IKK activation by IL-1β and TNFα. Moreover, mutations in the D. melanogaster homologue of TAB2 led to severe defects in antibacterial responses (D. Ferrandon, personal communication). Interestingly, most of these mutations have been mapped to the ubiquitin-binding domain of dTAB2, suggesting that ubiquitin-mediated activation of TAK1 and IKK is an evolutionarily conserved mechanism. The TAK1 kinase complex also contains TAB1; however, the role of TAB1 in the NF-κB pathway is not clear, as reconstitution experiments showed that TAB1 is dispensable for IKK activation in vitro11. In addition, there is no evidence that RNAi or genetic ablation of TAB1 impairs IKK activation57. Furthermore, there is no apparent homologue of TAB1 in the Drosophila genome. Thus, TAB1 may not be involved in NF-κB activation, but in the regulation of other pathways such as the Wnt or TGFβ pathway58,59. In fact, TAK1 was first isolated as a TGFβ-activated kinase, but its mechanism of activation in this pathway is poorly understood60.
While TAK1 and TAB2 clearly play a major role in innate immune responses in D. melanogaster, the phenotypes of dTAK1 and dTAB2 mutants are not as severe as those of dIKK mutants 53. Similarly, although RNAi against TAK1 or TAB2 and TAB3 severely impaired IKK activation, there is still residual IKK activity (approximately 20–30%). While some of this residual activity may be because depletion by RNAi is incomplete, it is also likely that there is another minor pathway contributing to IKK activation. One possible activator of IKK is p62, a UBA domain containing protein that binds to TRAF6 and RIP61–63. Mice lacking p62 are partially defective in prolonged NF-κB activation by RANK, a TNFR that activates IKK through TRAF6 in osteoclasts64. Interestingly, p62 forms a complex with an atypical PKC (ζPKC), representing potentially another example of a ubiquitin receptor-containing kinase complex similar to the TAB2/TAK1 complex. Another potential activator of IKK is MEKK3, as murine cells lacking MEKK3 are partially defective in NF-κB activation in response to certain stimuli65,66. It is also possible that the residual IKK activation does not require an upstream kinase, but is induced by the oligomerization of IKK itself. In this case, TRAF6 or TRAF2 and RIP may somehow interact with IKK/NEMO to induce its oligomerization, causing IKK to autophosphorylate itself, resulting in its activation.
Recent studies have revealed that ubiquitin signaling plays an important role in adaptive immunity, particularly T cell activation (Figure 4). Stimulation of T cell receptors (TCR) activates a tyrosine kinase cascade that subsequently activates the serine/threonine kinase PKCθ. PKCθ then induces the formation of a protein complex composed of CARMA1, BCL10 and MALT1, which are recruited to the lipid rafts where TCR resides67,68. Two studies have shown that BCL10 and MALT1 induce the K63-linked polyubiquitination of NEMO by Ubc13/Uev1A, leading to IKK activation69,70. However, there is a discrepancy concerning the E3 ubiquitin ligase for NEMO between these studies. In one study, MALT1 directly ubiquitinates NEMO at K399, despite the fact that MALT1 does not contain any known E3 domain69. In the other study, MALT1 was found to recruit TRAF6 via its TRAF6-binding sites, and TRAF6, in turn ubiquitinates NEMO70. The latter study showed that MALT1 also induces auto-ubiquitination of TRAF6, which then recruits the TAK1/TAB2 complex to activate IKK. This entire process from MALT1-mediated polyubiquitination to IκB phosphorylation was reconstituted in vitro using purified recombinant proteins70. TRAF6 and TRAF2 are redundant in the TCR pathway, as RNAi of both TRAF2 and TRAF6 effectively blocked IKK activation and IL-2 production by TCR stimulation, whereas RNAi of either TRAF alone was less effective70. However, it remains to be seen whether TRAF2 and TRAF6 are required for T cell activation in vivo.
NF-κB is regulated by a large variety of stimuli, including microbial pathogens and DNA damaging agents, which have recently been shown to activate IKK through a ubiquitin-dependent but degradation-independent mechanism. An elegant example was provided from the study of DNA damage-induced IKK activation71. DNA damaging agents, especially those that generate double strand breaks, such as topoisomerase inhibitors and gamma irradiation, can activate NF-κB in certain cancer cells. This DNA damage response, which requires the cytoplasmic IKK complex, may be responsible for the resistance of some cancer cells to chemotherapy. A mechanism by which the nuclear DNA damage signal is transduced to the cytoplasmic IKK complex has recently been uncovered (Figure 4)71. Upon DNA damage, a small fraction of nuclear NEMO is modified by the ubiquitin-like protein SUMO, which further retains NEMO in the nucleus. DNA damage also activates the kinase ATM, which phosphorylates NEMO. This phosphorylation leads to the replacement of SUMO by ubiquitin at two lysine residues (K277/K309) within NEMO. Ubiquitinated NEMO subsequently enters the cytoplasm, where it associates with the rest of the IKK complex, resulting in IKK activation.
Another recent study has provided evidence that K63 polyubiquitination may be involved in IKK activation by NOD1 and NOD2, two cytosolic receptors for intracellular bacteria72. Mutations in NOD2 have been closely linked to Crohn's disease, an inflammatory bowel disease. NOD1 and NOD2 activate IKK through RIP2, a RIP1-like kinase. Through a process that is still not well understood, NOD2 and RIP2 induce the K63-linked polyubiquitination of NEMO at K285, thereby activating IKK (Figure 4)73. Interestingly, the NOD2 mutations found in Crohn's disease patients impair NOD2's ability to activate NF-κB and to induce NEMO polyubiquitination in cell culture experiments73. However, mouse knock-in experiments show that the NOD2 mutations associated with the disease lead to NF-κB activation rather than inhibition74. Thus, the signaling mechanisms of NOD proteins and the potential role of ubiquitination in NOD signaling require further investigation.
The regulatory role of ubiquitin may not be limited to the NF-κB pathway. Indeed, TRAF6 ubiquitination has recently been found to be important for the activation of the transcription factor IRF775. IRF7 regulates the production of type-I interferons in response to stimulation of certain Toll-like receptors (TLR7, 8 & 9) by viral RNA or bacterial DNA. IRF7 is activated through its phosphorylation by an unknown kinase, which is in turn activated by the MyD88-IRAK4-TRAF6 complex (Figure 4). Recent studies have shown that MyD88 and TRAF6 bind to IRF7, inducing its phosphorylation and nuclear translocation. Interestingly, Ubc13 and TRAF6 ubiquitination are required for IRF7 activation75, suggesting that K63 polyubiquitination of as yet unidentified targets may be involved in the activation of an IRF7 kinase.
Like phosphorylation, ubiquitination is a reversible covalent modification. Two deubiquitinating enzymes (DUBs) have recently been shown to play an important role in suppressing NF-κB activation at a step upstream of IKK. One of these DUBs is the cylindromatosis tumor suppressor protein CYLD, which inhibits IKK activation by cleaving K63-linked polyubiquitin chains on several proteins including TRAF2, TRAF6 and NEMO76–78. Mutations within the DUB domains of CYLD, which are found in patients with cylindromas, lead to the enhanced activation of NF-κB, thereby contributing to the pathogenesis of the tumors. A recent study found that CYLD also inhibits JNK activation79, consistent with the idea that CYLD interferes with TRAF ubiquitination or TAK1 activation, which functions upstream of both IKK and JNK. However, this study found that CYLD inhibits IKK activation by IL-1β, LPS and CD40, but not TNFα, whereas previous studies showed that CYLD inhibits IKK activation by TNFα76–78. The reason for this discrepancy is not clear.
Another DUB that acts in this pathway is A20, an NF-κB induced protein that inhibit NF-κB in a negative feedback loop80–82. A20 deficient mice develop severe inflammatory diseases in multiple organs partly due to the enhanced and prolonged activation of NF-κB by proinflammatory stimuli including TNFα and LPS83. A20 contains a novel OTU type deubiquitination enzyme domain at the N-terminus, and several zinc finger domains in the remainder of the protein. The OTU domain disassembles K63-linked polyubiquitin chains from RIP (in the TNFα pathway)81 and TRAF6 (in the LPS pathway)80, thereby suppressing IKK activation. Moreover, A20 also functions as a ubiquitin ligase through its zinc finger domains to assemble K48-linked polyubiquitin chains on RIP after the K63-linked chains are removed by the OTU domain81. K48-linked polyubiquitination targets RIP for degradation by the proteasome, further diminishing IKK activation. Thus, A20 provides a dramatic example that a switch in polyubiquitin chains can profoundly influence the signaling and degradation of targets. A20 also inhibits the activation of the transcription factor IRF3 by RNA viruses84. In this case, the role of ubiquitin is less clear, as overexpression of an A20 mutant lacking the OTU domain still inhibits IRF3. It appears that A20 binds directly to and inhibits TBK1, an IKK-like kinase that phosphorylates IRF3.
The past decade has witnessed tremendous progress in elucidating the intertwining between ubiquitination and phosphorylation in the NF-κB pathway. It is now well established that phosphorylation is a prerequisite for ubiquitination of IκB and the NF-κB precursors p100 and p105. The identification of the ubiquitination machinery, especially the ubiquitin ligase complex containing βTrCP, which binds specifically to phosphorylated substrates, provides the molecular basis for the signal-dependent degradation of IκB and processing of p100 and p105. Recent studies have also identified several ubiquitin pathway components that function upstream of IKK, including the TRAF proteins as E3s, Ubc13/Uev1A as E2s, TAB2 and TAB3 as ubiquitin receptors, and CYLD and A20 as DUBs (Table 1). These results lend strong support to the thesis that ubiquitination can also regulate phosphorylation by activating IKK through a proteasome-independent mechanism.
While the traditional role of ubiquitin in degrading and processing NF-κB inhibitors is widely accepted, there are several important issues that remain to be addressed. For example, what is the mechanism underlying the highly processive polymerization of ubiquitin to form polyubiquitin chains? How are different E2 selected to a common SCF ubiquitin ligase scaffold to ubiquitinate distinct substrates? Is the assembly of a specific SCF complex regulated by the availability of substrates? How is ubiquitinated IκB, but not NF-κB present within the same complex, selectively degraded by the proteasome? How do p100 and p105 escape complete degradation by the proteasome? Are there additional proteins that escort the ubiquitinated IκB or p105/p100 to the proteasome?
Much also remains to be learnt about the non-traditional role of ubiquitin in the NF-κB pathway. Specifically, how does binding of a ubiquitinated protein (e.g, TRAF6 or RIP) to a ubiquitin receptor (e.g, TAB2 or TAB3) activate the receptor-associated kinase (e.g. TAK1)? What is the function of Nemo ubiquitination? Does NIK activation in the non-canonical pathway of NF-κB activation involve TRAF proteins and/or ubiquitin? Are there other NF-κB activation pathways that utilize ubiquitin as a signaling molecule for IKK activation? Are there other E2s (e.g., Ubc4/5) and E3s that may regulate IKK? In more general terms, is ubiquitin signaling involved in activating other enzymes including other kinases and ATPases? In this regard, the 19S proteasome contains multiple ATPases and ubiquitin binding proteins. Does the binding of polyubiquitinated protein substrates to the 19S proteasome activate its associated ATPase activity? If so, ubiquitin regulation of enzymes may be a unified mechanism underlying both the traditional and non-traditional functions of this protein tag.
Research in the Chen laboratory is supported by grants from NIH (R01-GM63692), the Welch Foundation (I1389) and American Cancer Society (RSG0219501TBE). Z.J.C is a Leukemia and Lymphoma Society Scholar and a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious Diseases.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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SHANGHAI During the eight years it took Dai Congrong to translate "Finnegans Wake" into Chinese, she read numerous versions of the dictionary, took voluminous notes, and fought off despair and opposition from her husband.
But her work on the first Chinese translation of the complex, stream-of-consciousness James Joyce opus about an Irish family paid off when the first print run of 8,000 sold out within weeks of hitting store shelves in December.
"It was dull and depressing during the first two years," the 41-year-old literature professor at Shanghai's Fudan University told Reuters. "I was also starting to have doubts about the project because I'd spent two years on the book but not a single word was translated."
A seasoned translator of books about culture and identity, Dai, who taught herself English, first started reading Finnegans Wake - a massive experimental novel notorious for its difficulty and convoluted prose - while at university.
Research into Joyce as part of her doctorate followed, and though her academic advisor recommended Dai translate the book when she successfully defended her dissertation, its difficulty scared her off. A later invitation from a publisher she knew, though, proved irresistible, and she began.
Joyce's frequent use of made-up words sent her poring over multiple versions of English dictionaries, making notes on every page of the book. Two years of preparation were followed by six of actual translation - in between teaching classes at the university as well as fulfilling other academic duties.
There were also obstacles at home.
"I have to take care of my young son. What's more, my family - especially my husband - didn't support the project," Dai said.
"He thought that despite the time and energy I needed to put into it, it would not make much money because the book is too difficult for an average person."
Despite everything, Dai found satisfaction in her labors.
"I enjoyed the moment when I finally found the exact and appropriate translation of a word or a sentence. I now know more about Joyce and the Irish culture," she said. "I think Joyce as a person was difficult to get along with for ordinary people, but I think he is a great person and a talented one."
Microbloggers on Sina Weibo, the Chinese equivalent of Twitter, lavished more praise on Dai's achievement than they did on the book itself.
"Oh my God! How admirable Dai Congrong is! How was she able to continue translating one book for eight years?" wrote Yao Zhenghua.
When her publisher told her that the 755-page book had sold out in three weeks, she was pleased but astonished.
"Actually, the news was so unbelievable that I first thought they were wrong," she said. "It shows our people are thirsty for culture and knowledge."
Dai's toil remains far from over, since she has actually translated only the first third of the book. Two more volumes are planned.
"Even I don't know how long it will take to finish," she said. "But I will do my best."
(Reporting by Shanghai newsroom and Kazunori Takada, writing by Elaine Lies, editing by Paul Casciato)
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subset: commoncrawl


text: 136 NOUS43 KPAH 152056 PNSPAH ILZ075>078-080>094-INZ081-082-085>088-KYZ001>022-MOZ076-086-087- 100-107>112-114-160900- Public Information Statement National Weather Service Paducah, KY 356 PM CDT FRI JUL 15 2016 ...Safety Rules for Dangerous Heat across our region... High Pressure is expected to develop and expand across the Quad State region starting late this weekend and persist through most of the next week. Although some locations may see brief relief from rain early next week...most areas will see a prolonged and elevated period of heat and humidity...especially in Southeast Missouri. Heat is the number one weather related killer in the United States, claiming some 350 lives each year. To ensure that you and your loved ones are not a victim of the deadly heat, here are some heat safety tips for you to follow. Remain in air conditioned places as much as possible. Eliminate, reduce, or reschedule all outdoor activities to the coolest time of the day. The hours of 11 am to 7 pm usually yield the highest heat index values. However, the intensity of the sun, even in the morning, should be taken into consideration. Full sunshine can increase heat index values by up to 15 degrees. If you must be outdoors, take frequent breaks in the shade. Dress for the heat by wearing lightweight, light colored clothing. Wear a high S P F sun screen and wear a hat to protect your face and head. Drink plenty of water or non-alcoholic sport drinks, even if you do not feel thirsty. Eat frequent, but small, lower protein meals, such as fruits, vegetables, and salads. Frequently check on the elderly, the very young, and those individuals with chronic health problems, as they are more susceptible to the dangers of heat. Contact your local government or community agency to find air conditioned locations to stay in if your do not have immediate access to an air conditioned place during the hottest part of the day. Never leave persons or animals in a closed area exposed to the sun, such as a vehicle. Temperatures in these places can become deadly in a matter of minutes. If live stock or pets must remain outdoors, try to make sure that they are provided with plenty of cool water and shade, whenever possible. Remember that the dangers of heat are cumulative, so it is especially important that you follow these safety rules for prolonged heat waves. Recognize and treat symptoms of heat disorders, such as heat cramps, heat exhaustion, and heat stroke quickly. Seek immediate medical attention or call 9 1 1 , especially if symptoms of heat exhaustion or heat stroke are observed. These include, but are not limited to vomiting, dizziness and exhaustion, mental confusion, headache, rapid weak pulse or breathing, dry red skin and high body temperature. Heat index values around 105 degrees or higher make heat cramps or heat exhaustion likely, and heat stroke possible with prolonged exposure and physical activity. $$ Smith


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From what i can tell Rode NTG2 sound good BUT the build quality is atrocious. 2 mics felt apart on me. Ss any recommendations for decent sounding mic07-20-2016, 02:03 PM by GaryNattrass
Anyone uses the Audio-Technica BP 4029?Thread Starter: MartinF
Hi All,Yesterday, 04:49 AM by MartinF
I am preparing for a documentary shoot, very verite and I will be shooting alone, so no sound recordist. As recording audio is going
The right steadicam for the budgetThread Starter: David Evans
Hi!07-21-2016, 12:00 PM by David Evans
I currently own a merlin steadicam which seemed pretty good when i bought it (and probably is), but its neverending options to fine balace
Dictation microphone for noisy environmentThread Starter: linuxizer
This is definitely not a movie question, but similar to recording live productions I think, and given that I'm a member here I was hoping someone might07-24-2016, 03:40 AM by linuxizer
Zoom F8 White Screen* need help*Thread Starter: rustyrehl
The exact same thing is happening to me that happened to this guy: https://www.reddit.com/r/LocationSou...oubleshooting/07-21-2016, 07:29 PM by scorsesefan
Pecluliar Effect - What is it?Thread Starter: Paul F
Please listen to this clip from 6:40 to 7:00. On my laptop, the clip between those two points does something funny to my ears. What is this and why does07-23-2016, 10:10 AM by Alex H.


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Urban forests have more benefits than raising a neighborhood's property value; the trees minimize pollution and soothe our minds after a long day.
From "Better Living through Forestry" by Sam Boykin. Reprinted with permission from the September/October 2003 issue of Sierra.
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text:
Stands for 'typical bloody SITA': the Head of Marketing in floods of tears after the CEO has slept through a presentation; backbiting colleagues; bewildering delays in payments; world hunger.
'Have the invoices been paid?'
'I know it's three months overdue, but it's TBS I'm afraid.'
'I know it's three months overdue, but it's TBS I'm afraid.'
by Uncle Whippity January 12, 2004
Trending Now
Top Definition
Tech Bitch Syndrom---Typically used on girl from Georgia Tech but can be substituted for any school with a larger male population. The girl, commonly uncool when surround by girls from other schools, becomes a bitch when many desperate college guys realize that is all they have.
That girl has major TBS, she took my alcohol and left.
Jenny Long
Jenny Long
by Anonymous September 23, 2003
True Brother Status
#TBS, For when a fraternity brother has your back and earns the status of a true brother. This can mean they helped you out when you were in some kind of need.
by RX_King May 23, 2015
the back seat of a car where two people would have sex.
let's take this to TBS so we can fuck.
by bfeezymack June 22, 2011
To Be Seen... A female chav hangs around with teenage boys to make themselves look good. Slags of all her friends and wears big cheap gold earings. Commonly waers silly mini skirt and stupid FM boots. Oh and dont forget the mountians of makeup
Just watch Little Britian... you'll see.
Or go to any major shopping centre... always plenty there!
Or go to any major shopping centre... always plenty there!
by XentriX February 09, 2005
Atlanta independent television station (actually it is WTBS-17 in Atlanta and TBS Superstation outside Atlanta), owned by Time Warner.
TBS stands for Turner Broadcast System
by James Westerfield January 03, 2004
Turn-based strategy (game)
Civilization is a TBS game, and Age of Empires is a RTS (real time strategy) game.
by Nomyd March 14, 2012


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MCTS in Windows Presentation Foundation since nov 18th 2009
Employed by Bridge Systems BV since may 2010
MCTS in Net 4.0 Data Access since sept 2012
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/14/2014 8,270,475 931,289 8.880675
1/31/2014 7,672,834 1,068,709 7.179535
Short Interest for Feb 28 is released. It is 8,420,000. Up a little from the end of January, but in line with the number of short shares listed for October, November, and December.
Short sellers are not driving the high share volume in ELNK for the past 3 months.
No worries.
I agree that the change in short interest over the past few months is minor. I think that this data indicates that the Shorts are not the main controlling factor.
I posted earlier that there are 8 institutions with large holdings in ELNK. It seems that they are trading between themselves. The share price may have more to do with tax loss selling than anything else.
So just on yahoo which these #s are pretty close... lets look at it for a moment per below and here on yahoo in key stats. So about 88% is held by institution/insiders that leaves 12% of the stock to trade hands by investors or commoners and 7.6% or over half of the shares able to trade hands are being shorted on this thinly traded stock, but u think they have no influence on the stock.
Food for thought....
% Held by Insiders1: 1.62%
% Held by Institutions1: 85.90%
Short % of Float (as of Jan 31, 2014)3: 7.60%


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Yesterday, 08:23 PMlatest patch? Sorry for being a bit tardy - end of the school term means my work is pretty intense for a bit. I'll get the sign ups ready soon...128 replies | 2866 view(s)
23-07-2016, 09:42 PMEthelred replied to a thread Total Warhammer in PC Gaming DiscussionIt's DLC I bought but then had some kids and never got round to it. Still on my backlog (with Skyrim). I have heard good things though. There...293 replies | 28358 view(s)
23-07-2016, 09:27 AMEthelred replied to a thread Total Warhammer in PC Gaming DiscussionI think maybe something based on Men of War or similar (dawn of war even?) would be better. The engine wouldn't cope very well with small squad...293 replies | 28358 view(s)
23-07-2016, 09:23 AMI'm afraid I don't agree. At lower levels AI & vs poor cmdrs maybe, but once you start coming across pilots with chaff, gimbals aren't so good. ...4528 replies | 524378 view(s)
21-07-2016, 02:42 PMI'd always try and slot in resting teams back where they were if possible. Going back to older teams I'd have to look at on a case by case -...128 replies | 2866 view(s)
19-07-2016, 06:20 PMOK. I'll extend the games that need extending that will take us to the end of the month. The next season for the DoD will start mid August. ...128 replies | 2866 view(s)
18-07-2016, 07:03 PMI'd pay money to see you frustrated by voice attack I think :-) But, there's nothing stopping you setting it up so the voice commands are in any...4528 replies | 524378 view(s)
18-07-2016, 07:30 AMIf you two can agree a date then I will extend it. Sign ups will go up this week with timeline. in fact - any mileage for an extended break...128 replies | 2866 view(s)
18-07-2016, 07:29 AMIs voice attack not an option? i run a Hotas and using voice attack to control power allocation, jump drive etc means I don't have to take my...4528 replies | 524378 view(s)
15-07-2016, 05:22 PMHow long does he need it for?128 replies | 2866 view(s)
15-07-2016, 03:29 PMMaybe we'll have to wait for ageing to have an effect to knock Grinn off the top spot? good work Grinn - looks like we need some fresh blood up...128 replies | 2866 view(s)
15-07-2016, 09:59 AMEthelred replied to a thread A fact indeed: UK votes to leave EU [was: any facts on the EU ref?] in Other StuffI'm a lifelong hand-wringing looney lefty and the problem with Corbyn is that he speaks to the majority of Left-wing Labour members. That is...1022 replies | 30279 view(s)
15-07-2016, 08:45 AMThanks to EU rules, all modern electrics have the same standard so you won't have any problems.128 replies | 2866 view(s)
14-07-2016, 10:50 PMEthelred replied to a thread NES Classic Edition in Other StuffKinda like a steam summer sale, something I'd spend £ on to play maybe once, then it would sit under the telly gathering dust. Like my Wii.24 replies | 518 view(s)
13-07-2016, 07:47 PMif you "low wake" - normal flight -> Supercruise - your FSD can be blocked by the mass of nearby ships causing a delay in spool up. if you "high...4528 replies | 524378 view(s)
13-07-2016, 06:32 PMThere is a "most hostile ship" key bind that automatically targets the highest threat to you and you should then be able to see what type of ship it...4528 replies | 524378 view(s)
12-07-2016, 10:25 AMOutstanding game all rolled - thankfully no admin results needed! MD3 is go deadline (soft because of holidays) 22nd July. Let me know in this...128 replies | 2866 view(s)
11-07-2016, 11:20 PMFor next season, if I can get some help, we could publish the fixture list with a link to the thread? I could announce the MDs on the steam group...128 replies | 2866 view(s)
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