Activation of NF-B transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradaNF-B transcription factor is regulated by IB proteins of which IB␣ is the main and best characterized member (1-3). Proteasome-mediated degradation of IB␣ releases NF-B and allows its localization in the nucleus (4 -9). Phosphorylation of Ser 32 -Ser 36 residues and subsequent ubiquitination of IB␣ are prerequisites to make the protein susceptible to proteasome attack (10 -13). While the kinase complex accounting for IB␣ phosphorylation has been recently characterized (14 -20), factors necessary for ubiquitination and targeting of IB␣ to the proteasome remain unknown. Covalent attachment of polyubiquitin to substrate proteins involved a cascade of ubiquitin transfer reactions with E1, 1 a ubiquitin-activating enzyme, and E2 a ubiquitin-conjugating enzyme that operates in conjunction with a specificity factor E3 (21-26). It has been suggested that E3 functions in substrate recognition and E2 positioning. Although E2 enzymes belonging to the Ubc4/Ubc5 family can ubiquitinate IB␣ in vitro, the E3 responsible for the signalinduced ubiquitination of IB␣ remains to be identified (10,27). A novel class of E3 is represented by the Skp1-Cullin-F-box protein complexes (SCFs) (28 -33). The core component of these newly identified E3s is Skp1, which assembles with different F-box proteins and has been shown in human cells to interact selectively with CUL-1, but not with other Cullin proteins belonging to the Cdc53 family (34 -36). The role of the F-box proteins in these SCF complexes is to recruit phosphorylated substrate proteins to trigger their ubiquitination (28 -33). Like the other members of the F-box protein family, human TrCP, which we recently identified (37), has a modular organization with an F-box motif involved in proteasome targeting through interaction with Skp1, and a seven WD repeats binding domain for interaction with substrate proteins (see Fig. 1A). We hypothesized that TrCP could be the F-box adaptor protein allowing recruitment of IB␣ by a SCF E3 ubiquitin-protein ligase complex that ubiquitinates IB␣ and makes it a substrate for degradation by the proteasome. EXPERIMENTAL PROCEDURESCell Lines, Transfections, and Infections-Subconfluent cells of the 293 human embryo kidney cell line were transfected by Lipofect-AMINE TM Plus (Life Technologies, Inc.) with the indicated reporter plasmids and pcDNA3 vectors expressing TrCP proteins or with no TrCP insert. Fluorigenic substrate luciferin served to quantify luciferase reporter gene expression in cytoplasmic extracts obtained by lysis in phosphate buffer containing 1% Nonidet P-40. The pcDNA3-TrCP or -TrCP⌬F constructs are described in Ref. 37. TrCP and TrCP⌬F coding sequences were amplified by polymerase chain reaction and inserted in fusion with the Myc/His double tag in the pcDNA3.1 Myc/ HisA vector (Invitrogen). SV5-tagged wild type or SV5-tagged S32A/ S36A phosphorylation-deficient mutant IB␣ are described in Ref. 11, 3Enh-B-ConA and ConA...
Gliotoxin targets catalytic activities of the proteasome efficiently. Inhibition by gliotoxin may be countered by reducing agents, which are able to inactivate the disulfide bridge responsible for the inhibitory capacity of gliotoxin.
The Rel/NF-kB family of transcription factors controls the expression of a wide variety of genes that are implicated in immune and in¯ammatory responses and cellular proliferation. Disregulation of NF-kB is associated with cellular transformation and the maintenance of a high anti-apoptotic threshold in transformed cells. NF-kB activity is in turn regulated by its sequestration in the cytoplasm by the inhibitor IkB. IkBa, the most abundant and well-characterized member of the IkB multiprotein family, is rapidly degraded in response to multiple physiologic stimuli. In the present study we show that not only the amino-terminus, but also the carboxy-terminus of IkBa contain transferable signals that must be simultaneously present in an unrelated protein to render it susceptible to activation-induced, proteasome-mediated degradation. We show here that IkBa amino-terminal modi®cations occur independently of the carboxy-terminus. Moreover, we present evidence indicating a critical role for the carboxy-terminal region in facilitating proteolysis by the catalytic core of the proteasome. When incubated with 20S proteasome extracted from rat liver, IkBa was quickly degraded while a deletion mutant lacking the carboxy-terminus was resistant to proteolysis. Likewise, chimeric proteins of beta-galactosidase with the IkBa carboxy-terminus were degraded in vitro independently of the presence of the IkBa amino-terminus, whereas chimeric proteins lacking the IkBa carboxy-terminus were stable. Our results identify the carboxy-terminus of IkBa as a domain critical for degradation through interaction with an as yet unidenti®ed component of the proteasome.
Signal-induced phosphorylation and ubiquitination of IB␣ targets this inhibitor of NF-B for proteasome-mediated degradation, thus permitting the release of active NF-B. Upon cell stimulation, NF-B activation results in neotranscription and neosynthesis of its own inhibitor, IB␣. As reported earlier, the neosynthesized inhibitor is then accumulated in the nucleus, where it rapidly binds to and terminates the function of nuclear NF-B upon withdrawal of the stimulus. The present work was aimed at understanding how NF-B activity is preserved while stimuli persist, despite intense, simultaneous IB␣ neosynthesis, which would be expected to end NF-B activity. We here show that incoming IB␣ in the nucleus represents a target for resident nuclear proteasome complexes. Signal-induced, proteasome-dependent degradation of phosphorylated and ubiquitinated IB␣ occurs in the nucleus, thus permitting the onset and persistence of NF-B activity as long as stimulation is maintained. Our results suggest that intranuclear proteolysis of IB␣ is necessarily required to avoid self-termination of NF-B activity during cell activation.Inhibitory IB proteins tightly control the biological activity of Rel/NF-B transcription factors through their association with homo-or heterodimers of this family. Members of the family share a highly conserved NH 2 -terminal sequence termed the Rel homology domain, which is required for DNA binding, dimerization, nuclear localization, and interaction with the IB molecules. In response to an inflammatory stimulus, cytokine, or viral infection, IB proteins are rapidly degraded by the 26 S multicatalytic proteasome. Degradation of IB␣, the most intensively characterized inhibitor, requires phosphorylation on serine residues 32-36 (1-6) by the activated IB kinase complex (reviewed in Ref. 7). This modification triggers recognition of IB␣ by the F-box/WD -TrCP protein, the receptor of the SCF E3 ubiquitin ligase, which marks IB␣ for ubiquitin-mediated proteolysis (8 -13). As a consequence of IB␣ degradation, the freed NF-B accumulates in the nucleus, where it activates gene transcription. NF-B acts on genes coding for cytokines, chemokines, immune receptors, and adhesion molecules, and its activation leads to a coordinated increase in the expression of inflammatory and immune response mediators (reviewed in Ref. 14).Apart from the well characterized inhibitory function on NF-B in the cytoplasm, IB␣ also participates in the inhibition of NF-B-dependent transcription in the cell nucleus. Once the stimulus is withdrawn, NF-B activity is rapidly shut down, ensuring that the B-dependent transcriptional activity is only transient (15,16). This is accounted for by two mechanisms. First, free, non-NF-B-associated IB␣ has the capacity to enter the nucleus when the protein is overexpressed from a heterologous promoter (17, 18). Such a property seems to rely on an active process mediated by a non-canonical nuclear import sequence located within the second ankyrin domain of IB␣ protein (19,20). Second, IB␣ has the ability...
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