Juan Qian

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.

Ligand-Stimulated Downregulation of the Alpha Interferon Receptor: Role of Protein Kinase D2§

ABSTRACT Alpha interferon (IFN-α) controls homeostasis of hematopoietic stem cells, regulates antiviral resistance, inhibits angiogenesis, and suppresses tumor growth. This cytokine is often used to treat cancers and chronic viral infections. The extent of cellular responses to IFN-α is limited by the IFN-induced ubiquitination and degradation of the IFN-α/β receptor chain 1 (IFNAR1) chain of the cognate receptor. IFNAR1 ubiquitination is facilitated by the βTrcp E3 ubiquitin ligase that is recruited to IFNAR1 upon its degron phosphorylation, which is induced by the ligand. Here we report identification of protein kinase D2 (PKD2) as a kinase that mediates the ligand-inducible phosphorylation of IFNAR1 degron and enables binding of βTrcp to the receptor. Treatment of cells with IFN-α induces catalytic activity of PKD2 and stimulates its interaction with IFNAR1. Expression and kinase activity of PKD2 are required for the ligand-inducible stimulation of IFNAR1 ubiquitination and endocytosis and for accelerated proteolytic turnover of IFNAR1. Furthermore, inhibition or knockdown of PKD2 robustly augments intracellular signaling induced by IFN-α and increases the efficacy of its antiviral effects. The mechanisms of the ligand-inducible elimination of IFNAR1 are discussed, along with the potential medical significance of this regulation.

Anti-tumorigenic effects of Type 1 interferon are subdued by integrated stress responses

Viral and pharmacological inducers of protein kinase RNA-activated (PKR)-like ER kinase (PERK) were shown to accelerate the phosphorylation-dependent degradation of the IFNAR1 chain of the Type 1 interferon (IFN) receptor and to limit cell sensitivity to IFN. Here we report that hypoxia can elicit these effects in a PERK-dependent manner. The altered fate of IFNAR1 affected by signaling downstream of PERK depends on phosphorylation of eIF2α (eukaryotic translational initiation factor 2-α) and ensuing activation of p38α kinase. Activators of other eIF2α kinases such as PKR or GCN2 (general control nonrepressed-2) are also capable of eliminating IFNAR1 and blunting IFN responses. Modulation of constitutive PKR activity in human breast cancer cells stabilizes IFNAR1 and sensitizes these cells to IFNAR1-dependent anti-tumorigenic effects. Although downregulation of IFNAR1 and impaired IFNAR1 signaling can be elicited in response to amino-acid deficit, the knockdown of GCN2 in melanoma cells reverses these phenotypes. We propose that, in cancer cells and the tumor microenvironment, activation of diverse eIF2α kinases followed by IFNAR1 downregulation enables multiple cellular components of tumor tissue to evade the direct and indirect anti-tumorigenic effects of Type 1 IFN.

Vascular endothelial growth factor-induced elimination of the type 1 interferon receptor is required for efficient angiogenesis.

Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) and antagonized by type 1 interferons, including IFN-α/β. On engaging their respective receptors (VEGFR2 and IFNAR), both stimuli activate protein kinase D2 (PKD2) and type 1 IFNs require PKD2 activation and recruitment to IFNAR1 to promote the phosphorylation-dependent ubiquitination, down-regulation, and degradation of the cognate receptor chain, IFNAR1. Data reveal that PKD2 activity is dispensable for VEGF-stimulated down-regulation of VEGFR2. Remarkably, VEGF treatment promotes the recruitment of PKD2 to IFNAR1 as well as ensuing phosphorylation, ubiquitination, and degradation of IFNAR1. In cells exposed to VEGF, phosphorylation-dependent degradation of IFNAR1 leads to an inhibition of type 1 IFN signaling and is required for efficient VEGF-stimulated angiogenesis. Importance of this mechanism for proangiogenic or antiangiogenic responses in cells exposed to counteracting stimuli and the potential medical significance of this regulation are discussed.

Inflammatory signaling compromises cell responses to interferon alpha

Interferon alpha (IFNα) is widely used for treatment of melanoma and certain other malignancies. This cytokine as well as the related IFNβ exerts potent anti-tumorigenic effects; however, their efficacy in patients is often suboptimal. Here, we report that inflammatory signaling impedes the effects of IFNα/β. Melanoma cells can secrete pro-inflammatory cytokines that inhibit cellular responses to IFNα/β via activating the ligand-independent pathway for the phosphorylation and subsequent ubiquitination and accelerated degradation of the IFNAR1 chain of type I IFN receptor. Catalytic activity of the p38 protein kinase was required for IFNAR1 downregulation and inhibition of IFNα/β signaling induced by proinflammatory cytokines such as interleukin 1 (IL-1). Activation of p38 kinase inversely correlated with protein levels of IFNAR1 in clinical melanoma specimens. Inhibition of p38 kinase augmented the inhibitory effects of IFNα/β on cell viability and growth in vitro and in vivo. The roles of inflammation and p38 protein kinase in regulating cellular responses to IFNα/β in normal and tumor cells are discussed.

Pathogen recognition receptor signaling accelerates phosphorylation-dependent degradation of IFNAR1.

An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in hosts defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity.

Role of p38 Protein Kinase in the Ligand-independent Ubiquitination and Down-regulation of the IFNAR1 Chain of Type I Interferon Receptor

Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of type I interferon (IFN) receptor is a robust and specific mechanism that limits the magnitude and duration of IFNα/β signaling. Besides the ligand-inducible IFNAR1 degradation, the existence of an “inside-out” signaling that accelerates IFNAR1 turnover in the cells undergoing the endoplasmic reticulum (ER) stress and activated unfolded protein responses has been recently described. The latter pathway does not require either presence of ligands (IFNα/β) or catalytic activity of Janus kinases (JAK). Instead, this pathway relies on activation of the PKR-like ER kinase (PERK) and ensuing specific priming phosphorylation of IFNAR1. Here, we describe studies that identify the stress activated p38 protein kinase as an important regulator of IFNAR1 that acts downstream of PERK. Results of the experiments using pharmacologic p38 kinase inhibitors, RNA interference approach, and cells from p38α knock-out mice suggest that p38 kinase activity is required for priming phosphorylation of IFNAR1 in cells undergoing unfolded protein response. We further demonstrate an important role of p38 kinase in the ligand-independent stimulation of IFNAR1 ubiquitination and degradation and ensuing attenuation of IFNα/β signaling and anti-viral defenses. We discuss the distinct importance of p38 kinase in regulating the overall responses to type I IFN in cells that have been already exposed to IFNα/β versus those cells that are yet to encounter these cytokines.

Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

Type 1 interferons (including IFNα/β) activate their cell surface receptor to induce the intracellular signal transduction pathways that play an important role in host defenses against infectious agents and tumors. The extent of cellular responses to IFNα is limited by several important mechanisms including the ligand-stimulated and specific serine phosphorylation-dependent degradation of the IFNAR1 chain of Type 1 IFN receptor. Previous studies revealed that acceleration of IFNAR1 degradation upon IFN stimulation requires activities of tyrosine kinase TYK2 and serine/threonine protein kinase D2 (PKD2), whose recruitment to IFNAR1 is also induced by the ligand. Here we report that activation of PKD2 by IFNα (but not its recruitment to the receptor) depends on TYK2 catalytic activity. PKD2 undergoes IFNα-inducible tyrosine phosphorylation on specific phospho-acceptor site (Tyr-438) within the plekstrin homology domain. Activated TYK2 is capable of facilitating this phosphorylation in vitro. Tyrosine phosphorylation of PKD2 is required for IFNα-stimulated activation of this kinase as well as for efficient serine phosphorylation and degradation of IFNAR1 and ensuing restriction of the extent of cellular responses to IFNα.

Melanoma cell‐secreted soluble factor that stimulates ubiquitination and degradation of the interferon alpha receptor and attenuates its signaling

Dear Sir, Adjuvant therapy of malignant melanoma with recombinant interferon alpha (IFN-α) is one of the few Federal Drug Administration (FDA)-approved treatments (Ascierto and Kirkwood, 2008). Although IFN-α exhibits potent anti-proliferative and anti-survival effects against tumor cells and endothelial cells of tumor vasculature in vitro, its therapeutic efficacy is limited (Mocellin et al., 2010). Overcoming these limitations requires a better understanding of the mechanisms that temper the effects of pharmaceutical IFN-α in the patient settings. All effects of IFN-α/β on cells are elicited through interaction with a cognate cell surface Type I IFN receptor that consists of IFNAR1 and IFNAR2c chains. This interaction leads to catalytic activation of Janus kinases (JAK), tyrosine phosphorylation of Signal Transducers and Activators of Transcription 1/2 (Stat1/2) proteins and activation of transcription of IFN-stimulated genes whose products restrict cell proliferation and viability (Platanias, 2005). Upon completion of the transcriptional program, the expression of negative regulators of Jak and Stat activities limits the magnitude and duration of this signaling. However, much earlier on, the ability of cells to further react to additional IFN-α/β molecules is rapidly eliminated by the ligand-induced downregulation of Type I IFN receptors from the cell surface (Coccia et al., 2006). IFN-α/β-induced downregulation of Type I IFN receptor is governed by ubiquitination of its IFNAR1 chain, mediated by the SCFβTrcp E3 ubiquitin ligase (Kumar et al., 2003). This ligase is recruited to IFNAR1 upon IFN-α/β-induced and Jak kinase activity-dependent phosphorylation of the Ser residues (e.g. Ser535) within the degron of IFNAR1 (Kumar et al., 2004). The manner in which levels of βTrcp2 are induced by mitogenic stimuli depends on the mitogen-activated protein kinase Erk1/2 (Spiegelman et al., 2002). A constitutively active BRAF mutant signals via Erk1/2 to increase the levels of βTrcp2, to downregulate IFNAR1, and to decrease cell responses to IFN-α in human melanoma cells (Kumar et al., 2007). Given that melanomas are known to secrete growth factors whose autocrine effect can also contribute to MAPK activation (Shih and Herlyn, 1994), we sought to determine whether pre-incubation of normal human melanocytes with media conditioned by 1205 Lu metastatic melanoma cells alters the levels of βTrcp and cellular responses to IFN-α. Pretreatment for only 2 h was not sufficient to induce βTrcp in melanocytes (Figure 1A). Surprisingly, activation of Stat1 in these cells was markedly inhibited. Similar observations could be made using media conditioned by WM278 and WM793 cells (data not shown). Conversely, when pre-treated with media from melanocytes, melanoma cells exhibited a greater extent of response to IFN-α without dramatic changes in βTrcp levels (Figure 1A). These data suggest either that normal melanocytes secrete a soluble factor that stimulates IFN-α signaling, or that melanoma cells produce an inhibitory factor. Furthermore, it is unlikely that either of these putative factors acts via changing the levels of βTrcp. Figure 1 (A) Melanocytes (MC) or 1205 Lu metastatic melanoma cells were pretreated with conditioned media from indicated sources for 2 h and then stimulated with IFN-α (30 IU/ml) for 30 min. Activation of STAT1 was monitored by immunoblot (IB) using pSTAT1 ... Further studies revealed that simply replacing conditioned media on melanoma cells with fresh media increased the extent of IFN-α signaling (data not shown). Furthermore, pretreatment with 1205 Lu metastatic melanoma cell-conditioned media (MM) robustly reduced the extent of Stat1 activation in response to IFN-α but not to IFN-β in normal human melanocytes (Figure 1B) and early stage radial growth phase WM115 melanoma cells or HeLa cells (data not shown). Given that, similarly to IFN-α, IFN-γ activates Janus kinases but acts via a completely different receptor (Platanias, 2005), it is plausible that a melanoma-secreted soluble factor acts by altering the levels of Type I IFN receptor available to interact with the ligand. Indeed, the cell surface levels of the IFNAR1 chain of this receptor were noticeably lower in 1205 Lu metastatic melanoma than in normal melanocytes or early WM115 cells that exhibited a robust response to IFN-α (Figure 1C). Low levels of IFNAR1 were reported to correlate with poor prognosis for patients with metastatic melanoma (Messina et al., 2008). These data suggest that downregulation of IFNAR1 may occur in metastatic melanomas. Downregulation of IFNAR1 is mediated by its βTrcp-dependent ubiquitination, which requires phosphorylation of IFNAR1 degron on Ser535 (Kumar et al., 2004). This phosphorylation was indeed increased in endogenous IFNAR1 from either HeLa or 293T cells treated with IFN-α or with melanoma cell-conditioned media (Figure 1D and data not shown). Pretreatment of these cells with Jak inhibitor I noticeably decreased Ser535 the phosphorylation of IFNAR1 induced by its natural ligand (i.e. IFN-α) but not by the conditioned melanoma media (Figure 1D). This indicates that soluble factor(s) secreted by melanoma cells can activate the ligand/JAK-independent pathway of IFNAR1 phosphorylation. We previously reported that this pathway involves priming phosphorylation of IFNAR1 on Ser532 (Bhattacharya et al., 2010). Indeed, a robust stimulation of this priming phosphorylation was seen in cells treated with melanoma-conditioned media (Figure 1D). We then sought to investigate whether these soluble factors affect ubiquitination and degradation of IFNAR1. Treatment of HeLa cells with melanoma-conditioned media robustly increased the extent of ubiquitination of endogenous IFNAR1 (Figure 1E). A similar result was obtained when ubiquitination of exogenously expressed Flag-IFNAR1 was analyzed (data not shown). Furthermore, consistent with a key role of IFNAR1 ubiquitination in the degradation of this receptor (Kumar et al., 2003), we observed that conditioned media from malignant melanoma cells when added to HeLa cells led to a robust stimulation of the turnover of endogenous IFNAR1 (Figure 1F). Similar accelerated degradation was observed on exogenously expressed receptor (data not shown). Our results suggest that melanoma-secreted factor(s) accelerate IFNAR1 phosphorylation, ubiquitination, degradation and downregulation through a novel ligand/JAK-independent pathway. It is plausible that this mechanism might be at least in part responsible for impeding the cellular responses to IFN-α. Future identification of these soluble factors and subsequent delineation of the signaling pathways through which these factors elicit these changes may point to novel targets for improving the efficacy of IFN-α therapy in melanoma patients.