Ubiquitin Ligase β-TrCP Research Articles

Role of TAZ as Mediator of Wnt Signaling

Wnt growth factors are fundamental regulators of cell fate, but how the Wnt signal is translated into biological responses is incompletely understood. Here, we report that TAZ, a biologically potent transcriptional coactivator, serves as a downstream element of the Wnt/β-catenin cascade. This function of TAZ is independent from its well-established role as mediator of Hippo signaling. In the absence of Wnt activity, the components of the β-catenin destruction complex--APC, Axin, and GSK3--are also required to keep TAZ at low levels. TAZ degradation depends on phosphorylated β-catenin that bridges TAZ to its ubiquitin ligase β-TrCP. Upon Wnt signaling, escape of β-catenin from the destruction complex impairs TAZ degradation and leads to concomitant accumulation of β-catenin and TAZ. At the genome-wide level, a substantial portion of Wnt transcriptional responses is mediated by TAZ. TAZ activation is a general feature of Wnt signaling and is functionally relevant to mediate Wnt biological effects.

Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

Regulatory Effects of Programmed Cell Death 4 (PDCD4) Protein in Interferon (IFN)-Stimulated Gene Expression and Generation of Type I IFN Responses

The precise mechanisms by which the activation of interferon (IFN) receptors (IFNRs) ultimately controls mRNA translation of specific target genes to induce IFN-dependent biological responses remain ill defined. We provide evidence that IFN-α induces phosphorylation of programmed cell death 4 (PDCD4) protein on Ser67. This IFN-α-dependent phosphorylation is mediated by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) in a cell-type-specific manner. IFN-dependent phosphorylation of PDCD4 results in downregulation of PDCD4 protein levels as the phosphorylated form of PDCD4 interacts with the ubiquitin ligase β-TRCP (β-transducin repeat-containing protein) and undergoes degradation. This process facilitates IFN-induced eukaryotic translation initiation factor 4A (eIF4A) activity and binding to translation initiation factor eIF4G to promote mRNA translation. Our data establish that PDCD4 degradation ultimately facilitates expression of several ISG protein products that play important roles in the generation of IFN responses, including IFN-stimulated gene 15 (ISG15), p21(WAF1/CIP1), and Schlafen 5 (SLFN5). Moreover, engagement of the RSK/PDCD4 pathway by the type I IFNR is required for the suppressive effects of IFN-α on normal CD34(+) hematopoietic precursors and for antileukemic effects in vitro. Altogether, these findings provide evidence for a unique function of PDCD4 in the type I IFN system and indicate a key regulatory role for this protein in mRNA translation of ISGs and control of IFN responses.

MLN4924, An Investigational NAE Inhibitor, Suppresses AKT and mTOR Signaling Pathway Through up Regulating REDD1 in Human Myeloma Cells

Abstract 1870 Introduction:The development and survival of normal plasma cells as well as multiple myeloma cells depend on an elaborately regulated ubiquitin proteasome system (UPS). Proteasome inhibitors such as bortezomib have proved to be highly active in the treatment of multiple myeloma. MLN4924, a newly developed investigational NEDD8 activating enzyme (NAE) inhibitor, exhibits promising anti-tumor effect through both clinical and laboratory observation. We sought to evaluate the individual signaling effects of MLN4924 in multiple myeloma, with the intent of further understanding the mechanism of action and identifying potential combinations. Methods:Human myeloma cell lines (MM.1S, MM.1R and U266) were treated with increasing concentrations of MLN4924 for 24, 48 and 72 hrs respectively or concurrently with bortezomib. Cell viability (MTT), apoptosis, western blot, RT-qPCR and siRNA assays were used to identify the cellular and molecular sequelae of MLN4924 treatment. Results:Single agent studies demonstrate that MLN4924 induces cytotoxicity in all three MM cell lines. Cytotoxicity is associated with increased apoptosis and suppression of AKT and mTOR signaling pathway, as detected by FACS and western blot. MLN4924 suppresses protein turnover of Cullin-ring ligases substrates leading to stabilization of specific proteins, such as p27, CDT1, NRF2. We find that REDD1, a substrate of CUL4 A–DDB 1–ROC1–β-TRCP ubiquitin ligase and negative regulator of mTOR pathway, increases in as early as 2 hours when treated with MLN4924. Knock-down of REDD1 using siRNA alleviates MLN4924 induced AKT, mTOR signaling suppression as well as the growth inhibition, which suggests that MLN4924 inhibits AKT, mTOR signaling through upregulating REDD1 protein leading to cytotoxicity.Combining MLN4924 with bortezomib synergistically enhances the antitumor effect of MLN4924. Western blot confirms the development of significantly increased procaspase and PARP cleavage, as well as substantial down regulation of AKT and mTOR signaling. Neither IL-6 nor IGF-1 is able to abrogate these combination effects. When we silence REDD1 in the combination assay in MM.1R, we find less cell apoptosis and suppression of AKT, mTOR pathway, which further support that REDD1 is an important regulator for MLN4924 induced cytotoxicity in MMs. Conclusion:MLN4924 is a potent investigational antitumor compound suppressing mTOR signaling pathways in myeloma cell lines. Down-regulation of the mTOR signaling pathway is associated with increased expression of REDD1. Combination of MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MMs cell lines which can overcome the prosurvival effects of growth factor (IL-6 and IGF-1). These findings could positively impact clinical combination strategies. Disclosures:Kaufman:Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Onyx: Consultancy; Merck: Consultancy.

Signaling pathways activated by the phytochemical nordihydroguaiaretic acid contribute to a Keap1-independent regulation of Nrf2 stability: Role of glycogen synthase kinase-3

Defense against oxidative stress is executed by an antioxidant program that is tightly controlled by the transcription factor Nrf2. The stability of Nrf2 involves the interaction of two degradation domains, designated Neh2 and Neh6, with the E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, respectively. The regulation of Nrf2 through the Neh6 degron remains largely unexplored but requires GSK-3 to form a phosphodegron. In this study, the cancer-chemopreventive agent nordihydroguaiaretic acid (NDGA) increased the level of Nrf2 protein and expression of heme oxygenase-1 (HO-1) in kidney-derived LLC-PK1 and HEK293T cells and in wild-type mouse embryo fibroblasts (MEFs). However, NDGA did not induce HO-1 in Nrf2 −/− MEFs, indicating that Nrf2 is required for induction. The relevance of the Nrf2/HO-1 axis to antioxidant protection was further demonstrated by the finding that the HO-1 inhibitor stannous-mesoporphyrin abolished protection against hydrogen peroxide conferred by NDGA. NDGA increased Nrf2 and HO-1 protein levels in Keap1 −/− MEFs, implying that Keap1-independent mechanisms regulate Nrf2 stability. Mutants of the Neh2 or Nrh6 domain and chimeric proteins comprising cyan fluorescent protein fused to Neh2 and green fluorescent protein fused to Neh6 exhibited longer half-lives in the presence of NDGA, demonstrating that NDGA targets both the Neh2 and the Neh6 degrons. In common with other chemopreventive agents, NDGA activated the ERK1/2, p38, JNK, and PI3K pathways. By using selective kinase inhibitors we found that PI3K, JNK, and p38 were responsible for the stabilization of Nrf2 and induction of HO-1 by NDGA. To explain how NDGA might up-regulate Nrf2 in a Keap1-independent manner we explored the participation of GSK-3β because it controls the Neh6 phosphodegron. Importantly, NDGA caused inhibitory phosphorylation of GSK-3β at Ser9 and at Thr390, and this was associated with a substantial reduction in Neh6 phosphorylation. Our study demonstrates that NDGA activates Nrf2 through multiple signaling cascades and identifies GSK-3β as an integrator of these signaling pathways and a gatekeeper of Nrf2 stability at the level of the Neh6 phosphodegron.

BIMEL, an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation

BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

Role of β-TrCP ubiquitin ligase receptor in UVB mediated responses in skin

Skin cancers are the most common cancers in the United States. Exposure to UVB radiation is a major risk factor for skin cancer induction. SCF β-TrCP E3 ubiquitin ligase has been found to be involved in cell cycle, cell proliferation and transformation. Aberrant up-regulation of beta-transducin repeats-containing proteins (β-TrCP) is often found in cancer cell lines and primary tumors. We have previously demonstrated that β-TrCP2 is over-expressed in chemically induced mouse skin tumors [1]. Various cellular stress stimuli, including UVB, induce an increase in β-TrCP1 mRNA and protein levels in human cells [2]. We have previously shown that inhibition of β-TrCP function, by induction of dominant negative β-TrCP2 (β-TrCP2 ΔF), in vitro in hTERT immortalized normal keratinocytes, results in increase in UVB induced apoptosis [3]. We have generated transgenic mice with inducible, selective expression of dominant negative β-TrCP2 in epidermis with the Keratin 5 promoter (K5-rTA × TRE-HA-β-TrCP ΔF). Here we report that inhibition of β-TrCP function in mouse epidermis results in decrease in UVB-induced edema, hyperplasia, and inflammatory response and increment in UVB-induced apoptosis in skin. Our results suggest that β-TrCP may be an essential player in UVB induced responses in skin and can be a potential therapeutic target for skin cancer.

APC Is Essential for Targeting Phosphorylated β-Catenin to the SCF β-TrCP Ubiquitin Ligase

Ubiquitin-dependent proteolysis is an important mechanism that suppresses the beta-catenin transcription factor in cells without Wnt stimulation. A critical step in this regulatory pathway is to create a SCF(beta-TrCP) E3 ubiquitin ligase binding site for beta-catenin. Here we show that the SCF(beta-TrCP) binding site created by phosphorylation of beta-catenin is highly vulnerable to protein phosphatase 2A (PP2A) and must be protected by the adenomatous polyposis coli (APC) tumor suppressor protein. Specifically, phosphorylated beta-catenin associated with the wild-type APC protein is recruited to the SCF(beta-TrCP) complex, ubiquitin conjugated, and degraded. A mutation in APC that deprives this protective function exposes the N-terminal phosphorylated serine/threonine residues of beta-catenin to PP2A. Dephosphorylation at these residues by PP2A eliminates the SCF(beta-TrCP) recognition site and blocks beta-catenin ubiquitin conjugation. Thus, by acting to protect the E3 ligase binding site, APC ensures the ubiquitin conjugation of phosphorylated beta-catenin.

Structure of the Complex between Phosphorylated Substrates and the SCF β-TrCP Ubiquitin Ligase Receptor: A Combined NMR, Molecular Modeling, and Docking Approach

The binding of phosphorylated peptides to the receptor plays a major role in many basic cellular processes in a variety of pathological states. Human beta-TrCP is a key component of a recently characterized E3 ubiquitin ligase complex that regulates protein degradation through the ubiquitin-dependent proteasome pathway. Docking studies were carried out to explore the structural requirements for the beta-TrCP substrates. Docking studies were performed on the bound conformation of the phosphorylated peptides determined by NMR, whereas the beta-TrCP structure was derived by X-ray from Protein Data Bank. After the docking calculation, during which the peptides were conformationally restrained, the complex presented herein was analyzed in terms of ligand-protein interactions and properties of contacting surfaces. The structural requirements for phosphorylated substrates in interaction with beta-TrCP were explored and compared with experimental data from TRNOESY and STD NMR results. The analysis revealed that the bend of the peptide structures, which is indispensable for beta-TrCP recognition, aligns two charged phosphate groups and a central hydrophobic group in a favorable arrangement that leads to the burial of the peptide surface in the binding cleft upon complexation. Through docking simulations, we have identified different specific binding pockets of beta-TrCP according to the ligand in interaction. These data should be valuable in the rational design of a ligand to be used in therapeutic approaches.

Plk1- and β-TrCP–dependent degradation of Bora controls mitotic progression

Through a convergence of functional genomic and proteomic studies, we identify Bora as a previously unknown cell cycle protein that interacts with the Plk1 kinase and the SCF–β-TrCP ubiquitin ligase. We show that the Bora protein peaks in G2 and is degraded by proteasomes in mitosis. Proteolysis of Bora requires the Plk1 kinase activity and is mediated by SCF–β-TrCP. Plk1 phosphorylates a conserved DSGxxT degron in Bora and promotes its interaction with β-TrCP. Mutations in this degron stabilize Bora. Expression of a nondegradable Bora variant prolongs the metaphase and delays anaphase onset, indicating a physiological requirement of Bora degradation. Interestingly, the activity of Bora is also required for normal mitotic progression, as knockdown of Bora activates the spindle checkpoint and delays sister chromatid segregation. Mechanistically, Bora regulates spindle stability and microtubule polymerization and promotes tension across sister kinetochores during mitosis. We conclude that tight regulation of the Bora protein by its synthesis and degradation is critical for cell cycle progression.

Transfer-NMR and Docking Studies Identify the Binding of the Peptide Derived from Activating Transcription Factor 4 to Protein Ubiquitin Ligase β-TrCP. Competition STD-NMR with β-Catenin

ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.

Raf inhibitor stabilizes receptor for the type I interferon but inhibits its anti-proliferative effects in human malignant melanoma cells

Interferon alpha (IFNα) is widely used in treatment of malignant melanoma patients. This cytokine acts on cells by engaging Type I IFN receptor consisting of two subunits, (IFNAR1 and IFNAR2) followed by activation of Janus kinases (Jak). Levels of IFNAR1 (regulated via degradation mediated by the βTrcp E3 ubiquitin ligase) and IFNα signaling were reduced in 1205Lu melanoma cell line that harbors activated BRAF and exhibits high levels of βTrcp ubiquitin ligase. Expression of stabilized IFNAR1 in melanoma cells decreased their tumorigenicity. Furthermore, RNAi-mediated BRAF knockdown and pharmacologic inhibition of either Raf or MEK1 decreased levels of βTrcp and stabilized IFNAR1. However, despite causing stabilization of IFNAR1, Raf inhibitor BAY 43-9006 interfered with cellular responses to IFNα most likely due to its ability to directly inhibit Jak activity. We discuss the implications of this result for combination therapy with BAY 43-9006 and IFNα in melanoma patients.

Gli2 Is Targeted for Ubiquitination and Degradation by β-TrCP Ubiquitin Ligase

The Hedgehog (Hh) signaling pathway plays a crucial role in embryogenesis and has been linked to the development of several human malignancies. The transcription factor Gli2 plays a key role in the transduction of Hh signals by modulating transcription of some Hh target genes, yet the mechanisms that control Gli2 protein expression are largely unknown. Here we report that beta-transducin repeat-containing protein (beta-TrCP) E3 ubiquitin ligase is required for Gli2 degradation. beta-TrCP2 directly binds wild type Gli2 and promotes its ubiquitination. Single amino acid substitution in Gli2 putative binding site inhibits its interaction with beta-TrCP2, its ubiquitination, and stabilizes the Gli2 protein. Stable Gli2 mutant is expressed in higher levels and is more potent in the activation of Gli-dependent transcription as compared with wild type Gli2. We also found that GLI2 protein is expressed highly in prostate cancer cell lines and primary tumors, whereas the level of GLI2 mRNA is not appreciably different in normal and neoplastic prostate. These data identify beta-TrCP2 as a pivotal regulator of Gli2 expression and point to an important role for posttranslational modulation of GLI2 protein levels in Hh pathway-associated human prostate cancer.

Stabilization of Basally Translated NF-κB-inducing Kinase (NIK) Protein Functions as a Molecular Switch of Processing of NF-κB2 p100

The non-canonical pathway based on processing of NF-kappaB2 precursor protein p100 to generate p52 plays a critical role in controlling B cell function and lymphoid organogenesis. Activation of this unique pathway by extracellular stimuli requires NF-kappaB-inducing kinase (NIK) and de novo protein synthesis. However, how NIK is regulated is largely unknown. Here, we systematically analyzed NIK expression at different levels in the presence or absence of different NF-kappaB stimuli. We found that NIK mRNA is relatively abundant and undergoes constitutive protein synthesis in resting B cells. However, NIK protein is undetectable. Interestingly, protein expression of NIK is steadily induced by B cell-activating factor or CD40 ligand, two major physiological inducers of p100 processing, but not by mitogen phorbol 12-myristate 13-acetate/ionomycin or cytokine tumor necrosis factor alpha, two well known inducers of the canonical NF-kappaB signaling. Remarkably, both B cell-activating factor and CD40 ligand do not significantly induce expression of NIK at translational or transcriptional level but rather rescue the basally translated NIK protein from undergoing degradation. Furthermore, overexpressed or purified NIK protein triggers p100 processing in the presence of protein synthesis inhibitor. Taken together, these studies define one important mechanism of NIK regulation and the central role of NIK stabilization in the induction of p100 processing. These studies also provide the first evidence explaining why activation of the non-canonical NF-kappaB signaling is delayed and can be inhibited by protein synthesis inhibitor as well as why most classical NF-kappaB stimuli, including mitogens and tumor necrosis factor alpha, fail to induce p100 processing.

Essential Role of IκB Kinase α in the Constitutive Processing of NF-κB2 p100

Processing of NF-kappaB2 precursor protein p100 to generate p52 is tightly controlled, which is important for proper function of NF-kappaB. Accordingly, constitutive processing of p100, caused by the loss of its C-terminal processing inhibitory domain due to nfkappab2 gene rearrangements, is associated with the development of various lymphomas and leukemia. In contrast to the physiological processing of p100 triggered by NF-kappaB-inducing kinase (NIK) and its downstream kinase, IkappaB kinase alpha (IKKalpha), which requires the E3 ligase, beta-transducin repeat-containing protein (beta-TrCP), and occurs only in the cytoplasm, the constitutive processing of p100 is independent of beta-TrCP but rather is regulated by the nuclear shuttling of p100. Here, we show that constitutive processing of p100 also requires IKKalpha, but not IKKbeta (IkappaB kinase beta) or IKKgamma (IkappaB kinase gamma). It seems that NIK is also dispensable for this pathogenic processing of p100. These results demonstrate a general role of IKKalpha in p100 processing under both physiological and pathogenic conditions. Additionally, we find that IKKalpha is not required for the nuclear translocation of p100. Thus, these results also indicate that p100 nuclear translocation is not sufficient for the constitutive processing of p100.

Regulation of the Discs Large Tumor Suppressor by a Phosphorylation-dependent Interaction with the β-TrCP Ubiquitin Ligase Receptor

The discs large (hDlg) tumor suppressor is intimately involved in the control of cell contact, polarity, and proliferation by interacting with several components of the epithelial junctional complex and with the APC tumor suppressor protein. In epithelial cells, hDlg protein stability is regulated through the ubiquitin-proteasome pathway: hDlg is actively degraded in isolated cells, whereas it accumulates upon cell-cell contact. During neoplastic transformation of epithelial cells, loss of the differentiated morphology and progression toward a metastatic phenotype correlate with down-regulation of hDlg levels and loss of contact-dependent stabilization. Here we show that upon hyperphosphorylation, hDlg interacts with the beta-TrCP ubiquitin ligase receptor through a DSGLPS motif within its Src homology 3 domain. As a consequence, overexpression of beta-TrCP enhances ubiquitination of Dlg protein and decreases its stability, whereas a dominant negative beta-TrCP mutant inhibits this process. Furthermore, a mutant Dlg protein that is unable to bind beta-TrCP displays a higher protein stability and is insensitive to beta-TrCP. Using RNA interference, we also demonstrate that endogenous beta-TrCP regulates hDlg protein levels in epithelial cells. Finally, we show that beta-TrCP selectively induces the degradation of the membrane-cytoplasmic pool, without affecting the nuclear pool of hDlg.

Mechanisms of ubiquitin-mediated, limited processingof the NF-κB1 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-κB, 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 IκB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918–934, recruits the SCF β-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-κB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IκBγ). 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.