Although constitutive activation of beta-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the beta-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that beta-catenin stabilizes the mRNA encoding the F-box protein betaTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of beta-catenin/Tcf transcription factor. CRD-BP binds to the coding region of betaTrCP1 mRNA. Overexpression of CRD-BP stabilizes betaTrCP1 mRNA and elevates betaTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)(betaTrCP) E3 ubiquitin ligase and in accelerated turnover of its substrates including IkappaB and beta-catenin. CRD-BP is essential for the induction of both betaTrCP1 and c-Myc by beta-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active beta-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of betaTrCP1, in the activation of dimeric transcription factor NF-kappaB and in the suppression of apoptosis in these cancers.

Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of Type I interferon (IFN) receptor is regulated by two different pathways one of which is ligand-independent. We report that this pathway is activated by inducers of the endoplasmic reticulum (ER) stress, including viral infection, in a PERK-dependent manner. Upon infection, activation of this pathway promotes phosphorylation-dependent ubiquitination and degradation of IFNAR1, and specifically inhibits Type I IFN signaling and antiviral defenses. Either knock-in of an IFNAR1 mutant insensitive to virus-induced turnover or conditional knockout of PERK prevented ER stress- and virus-induced IFNAR1 degradation while restoring cellular responses to Type I IFN and resistance to viruses. The role of this novel mechanism in pathogenesis of viral infections and therapeutic approaches to their treatment is discussed.

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

Phosphorylation of the degron of the IFNAR1 chain of the type I interferon (IFN) receptor triggers ubiquitination and degradation of this receptor and, therefore, plays a crucial role in negative regulation of IFN-␣/␤ signaling. Besides the IFN-stimulated and Jak activity-dependent pathways, a basal ligand-independent phosphorylation of IFNAR1 has been described and implicated in downregulating IFNAR1 in response to virus-induced endoplasmic reticulum (ER) stress. Here we report purification and characterization of casein kinase 1␣ (CK1␣) as a bona fide major IFNAR1 kinase that confers basal turnover of IFNAR1 and cooperates with ER stress stimuli to mediate phosphorylation-dependent degradation of IFNAR1. Activity of CK1␣ was required for phosphorylation and downregulation of IFNAR1 in response to ER stress and viral infection. While many forms of CK1 were capable of phosphorylating IFNAR1 in vitro, human CK1␣ and L-CK1 produced by the protozoan Leishmania major were also capable of increasing IFNAR1 degron phosphorylation in cells. Expression of leishmania CK1 in mammalian cells stimulated the phosphorylation-dependent downregulation of IFNAR1 and attenuated its signaling. Infection of mammalian cells with L. major modestly decreased IFNAR1 levels and attenuated cellular responses to IFN-␣ in vitro. We propose a role for mammalian and parasite CK1 enzymes in regulating IFNAR1 stability and type I IFN signaling.Cytokines that belong to a superfamily of interferons (IFNs), including type I IFN (such as IFN-␤ and numerous species of IFN-␣) and type II interferon (IFN-␥), are important for efficient antiviral defense (40, 51). Type I IFNs signal via interacting with the heterodimeric receptor complex composed of two chains (IFNAR1 and IFNAR2); ligand binding activates receptor-associated members of the JAK family of tyrosine kinases, Jak1 and Tyk2. These kinases phosphorylate and activate the signal transducers and activators of transcription (STAT) proteins, which increase transcription of the IFN-induced genes whose products exert antiviral, immunomodulatory, and antiproliferative effects. While some of the IFN actions might proceed in a STAT-independent manner, all biological functions of IFN-␣/␤ reported to date rely on the function of the type I IFN receptor complex (reviewed in references 1, 54, and 55).The IFNAR1 subunit of this receptor is essential for IFN-␣/␤ signaling. Mice lacking IFNAR1 display a deficiency in antiviral responses (24,36) and an altered immune activation in response to a number of microbial agents (57). Intriguingly, these mice do not display an increased susceptibility to a number of protozoans, including Leishmania spp. (57), which is lethal in animals that lack responses to IFN-␥ (50). However, similar to IFN-␥, IFN-␣ also activates STAT1 and upregulates the inducible nitric oxide synthase, which is essential in the early defense against Leishmania (5), which by itself stimulates the production of type I IFN during the early infection stage (14).In wild-type animals, the levels of I...

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAFdependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor jB (NF-jB) transcription factor activation triggered by proteolysis of its inhibitor IjB. IjB degradation is dependent upon its phosphorylation by the IjB kinase (IKK) complex and subsequent ubiquitination facilitated by b-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF V600E exhibit enhanced b-Trcp expression, increased IKK activity and a concomitant increase in the rate of IjBa degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor or by selective knock-down of BRAF V600E expression by RNA interference in human melanoma cells leads to decreased IKK activity and b-Trcp expression, stabilization of IjB, inhibition of NF-jB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-jB activity and to increased survival of melanoma cells.