The Wnt pathway controls numerous developmental processes via the ␤-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of ␤-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic ␤-catenin associates with APC and axin and is continuously phosphorylated by GSK-3␤, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3␤ from phosphorylating ␤-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of ␤-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces ␤-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate ␤-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3␤ and is both necessary and sufficient to initiate the ␤-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway.

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

The nuclear translocation of NF-KcB follows the degradation of its inhibitor, IKBcx, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of IKcBa, either by treating cells with various reagents or by mutagenesis of certain putative IKcBai phosphorylation sites, abolishes the inducible degradation of IKcBX. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in IKcBGi degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of IKcBa recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pIlcBa, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible IcBa degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of IlcBa. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

Activation of the transcription factor NF-κB is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor IκB in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-κB, IκB is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of IκB and its subsequent degradation. The ligase recognition signal is functionally conserved between IκBα and IκBβ, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-κB activation in response to TNFα and the consequent expression of E-selectin, an NF-κB-dependent cell-adhesion molecule. Inhibition of NF-κB function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

NF-B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit IB from cytoplasmic NF-B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of IB in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-B activation is associated with IB degradation. In vivo phosphorylation of IB␣ was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of IB␣ and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible IB␣ phosphorylation was abolished by several distinct NF-B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of IB␣ phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of IB␣ but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated IB␣ remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-B activation was abolished. It appears that activation of NF-B requires degradation of IB␣ while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

␤-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF␤-TrCP ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IB␣ and ␤-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished by competition with a pIB␣ peptide, or by a specific point mutation in the E3RS WD region, indicating an E3-substrate-type interaction. However, unlike pIB␣, which is targeted by SCF ␤-TrCP for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCF ␤-TrCP , which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold, and efficacy of a specific protein-ubiquitin ligase.

The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway.

A radioimmunoassay (RIA) has been developed and used to determine the expression of LMP-a membrane protein encoded by the LT3 region of the Epstein-Barr virus (EBV) genome-in cell lines of various origins. The RIA was highly sensitive, specific and reproducible. All EBV-negative cell lines were LMP-negative and 18 of 21 EBV-carrying cells were LMP-positive. LMP concentrations varied widely, ranging approximately from less than 4 ng up to 650 ng/mg protein. In several instances comparisons were made between lymphoblastoid (LCLs) and Burkitt lymphoma (BL) cell lines (EBV-positive or EBV-converted sublines of originally EBV-negative BL) originating from the same patient. In all such cases LMP and LMP-specific mRNA levels were higher in the LCLs. Most of the LMP was found in the cytosol fraction, yet this fraction was negative in immunoblotting tests. However, antiserum preincubated with the cytosol lost its ability to react in immunoblotting with membrane LMP, indicating that the 2 LMP forms (membrane and cytosol) are completely cross-reactive.