K. G. Suresh Kumar

UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity

Interferons (IFNs) regulate diverse cellular functions through activation of the Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway. Lack of Ubp43, an IFN‐inducible ISG15 deconjugating enzyme, leads to IFN hypersensitivity in ubp43−/− mice, suggesting an important function of Ubp43 in downregulation of IFN responses. Here, we show that Ubp43 negatively regulates IFN signaling independent of its isopeptidase activity towards ISG15. Ubp43 functions specifically for type I IFN signaling by downregulating the JAK–STAT pathway at the level of the IFN receptor. Using molecular, biochemical, and genetic approaches, we demonstrate that Ubp43 specifically binds to the IFNAR2 receptor subunit and inhibits the activity of receptor‐associated JAK1 by blocking the interaction between JAK and the IFN receptor. These data implicate Ubp43 as a novel in vivo inhibitor of signal transduction pathways that are specifically triggered by type I IFN.

The many faces of β -TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer

Beta-transducin repeats-containing proteins (β-TrCP) serve as the substrate recognition subunits for the SCFβ-TrCP E3 ubiquitin ligases. These ligases ubiquitinate specifically phosphorylated substrates and play a pivotal role in the regulation of cell division and various signal transduction pathways, which, in turn, are essential for many aspects of tumorigenesis. We review the functions of the SCFβ-TrCP ligases in the light of their relevance to cell growth, survival and transformation. Mechanisms underlying β-TrCP regulation and their aberration in human and animal cancer as well as prospective of targeting β-TrCP as a means of anticancer therapy are also discussed.

SCFHOS ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-α receptor

Down‐regulation of activated signaling receptors in response to their ligands plays a key role in restricting the extent and duration of the signaling. Mechanisms underlying down‐regulation of the type I interferon receptor consisting of IFNAR1 and IFNAR2 subunits remain largely unknown. Here we show that IFNAR1 interacts with the Homolog of Slimb (HOS) F‐box protein in a phosphorylation‐dependent manner, and that this interaction is promoted by interferon α (IFNα). IFNAR1 is ubiquitinated by the Skp1‐Cullin1‐HOS‐Roc1 (SCFHOS) ubiquitin ligase in vitro. HOS expression and activities are required for IFNα‐stimulated ubiquitination of IFNAR1, endocytosis of the type I interferon receptor, down‐regulation of IFNAR1 levels, and IFNAR1 proteolysis via the lysosomal pathway. Furthermore, modulations of HOS activities affect the extent of Stat1 phosphorylation and Stat‐mediated transcriptional activities as well as the extent of antiproliferative effects of type I interferons. These findings characterize SCFHOS as an E3 ubiquitin ligase that is essential for ubiquitination, proteolysis and down‐regulation of IFNAR1, and implicate HOS in the regulation of cellular responses to IFNα.

Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

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.

Virus-induced unfolded protein response attenuates antiviral defenses via phosphorylation-dependent degradation of the type I interferon receptor.

Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of the type I interferon (IFN) receptor is regulated by two different pathways, one of which is ligand independent. We report that this ligand-independent pathway is activated by inducers of unfolded protein responses (UPR), including viral infection, and that such activation requires the endoplasmic reticulum-resident protein kinase PERK. Upon viral infection, activation of this pathway promotes phosphorylation-dependent ubiquitination and degradation of IFNAR1, specifically inhibiting type I IFN signaling and antiviral defenses. Knockin of an IFNAR1 mutant insensitive to virus-induced turnover or conditional knockout of PERK prevented IFNAR1 degradation, whether UPR-induced or virus-induced, and restored cellular responses to type I IFN and resistance to viruses. These data suggest that specific activation of the PERK component of UPR can favor viral replication. Interfering with PERK-dependent IFNAR1 degradation could therefore contribute to therapeutic strategies against viral infections.

Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase

ABSTRACT Ubiquitin-dependent degradation of hormone receptors is emerging as a key mechanism that regulates the magnitude and duration of hormonal effects on cells and tissues. The pituitary hormone prolactin (PRL) is involved in regulating cell differentiation, proliferation, and survival. PRL engages its receptor (PRLR) to initiate various signaling cascades, including the phosphorylation and activation of Stat5. We found that PRL promotes interaction between PRLR and the F-box protein β-TrCP2, which functions as a substrate recognition subunit of the SCFβ-TrCP E3 ubiquitin ligase. This interaction requires PRLR phosphorylation and the integrity of serine 349 within a conserved motif, which is similar to conserved motifs present in other substrates of SCFβ-TrCP. The PRLRS349A mutant is resistant to ubiquitination and is more stable than its wild-type counterpart. Phosphorylated PRLR undergoes ubiquitination by SCFβ-TrCP in vitro. Knockdown of β-TrCP expression inhibits the ubiquitination and degradation of PRLR and promotes PRL-dependent phosphorylation of Stat5 as well as Stat5-dependent transcription in cells. Furthermore, the activation of Stat5 and the stimulation of cell growth by PRL are augmented in cells expressing the PRLRS349A mutant. These data indicate that PRLR is a novel SCFβ-TrCP substrate and implicate β-TrCP as an important negative regulator of PRL signaling and cellular responses to this hormone.

Oncogenic BRAF regulates β-Trcp expression and NF-κB activity in human melanoma cells

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor κB (NF-κB) transcription factor activation triggered by proteolysis of its inhibitor IκB. IκB degradation is dependent upon its phosphorylation by the IκB kinase (IKK) complex and subsequent ubiquitination facilitated by β-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAFV600E exhibit enhanced β-Trcp expression, increased IKK activity and a concomitant increase in the rate of IκBα degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAFV600E expression by RNA interference in human melanoma cells leads to decreased IKK activity and β-Trcp expression, stabilization of IκB, inhibition of NF-κB 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-κB activity and to increased survival of melanoma cells.

TYK2 activity promotes ligand-induced IFNAR1 proteolysis

The type I IFNR (interferon receptor) is a heterodimer composed of two transmembrane chains, IFNAR1 (interferon-alpha receptor 1 subunit) and IFNAR2, which are associated with the tyrosine kinases Tyk2 and Jak1 (Janus kinase 1) respectively. Ligand-induced down-regulation of the type I IFNR is a major mechanism of negative regulation of cellular signalling and involves the internalization and lysosomal degradation of IFNAR1. IFNalpha promotes the phosphorylation of IFNAR1 on Ser535, followed by recruitment of the E3 ubiquitin ligase, beta-TrCP2 (beta-transducin repeats-containing protein 2), ubiquitination of IFNAR1 and proteolysis. The non-catalytic role of Tyk2 in sustaining the steady-state IFNAR1 level at the plasma membrane is well documented; however, little is known about the function of Tyk2 in the steps that precede and succeed serine phosphorylation and ubiquitination of IFNAR1 in response to ligand binding. In the present study, we show that catalytic activation of Tyk2 is not essential for IFNAR1 internalization, but is required for ligand-induced IFNAR1 serine phosphorylation, ubiquitination and efficient lysosomal proteolysis.

Mammalian casein kinase 1α and its leishmanial ortholog regulate stability of IFNAR1 and type I interferon signaling

ABSTRACT 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-α/β 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 1α (CK1α) 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 CK1α 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 CK1α 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-α in vitro. We propose a role for mammalian and parasite CK1 enzymes in regulating IFNAR1 stability and type I IFN signaling.

Ligand-independent pathway that controls stability of interferon alpha receptor.

Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.