Identification of a Lysosomal Pathway Regulating Degradation of the Bone Morphogenetic Protein Receptor Type II

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease.     

SIAH-1 interacts with the Kaposi's sarcoma-associated herpesvirus-encoded ORF45 protein and promotes its ubiquitylation and proteasomal degradation

Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection.     

Identification of phosphorylation sites on the E3 ubiquitin ligase UBR5/EDD

UBR5 (ubiquitin protein ligase E3 component n-recognin 5)/EDD (E3 ligase identified by differential display) is an E3 ubiquitin ligase that is a potential biomarker for poor prognosis for recurrent, platinum-resistant ovarian cancer. UBR5 has a role in the DNA damage response and many such proteins are regulated by phosphorylation. UBR5 is a 309 kDa nuclear phosphoprotein that we previously identified as a substrate of the MAP kinase ERK2. With its 477 potential phosphorylation sites, little is known about UBR5 phosphorylation and how it may regulate protein function. Currently, thirty-four sites of phosphorylation on UBR5 have been reported in the literature, mostly identified by large scale proteomics studies of tissues or of cells after various treatments; however, no studies have specifically targeted the identification of UBR5 phosphorylation sites. In this study, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain a total sequence coverage of 64.3% from combining tryptic and GluC digests on UBR5 isolated from transfected COS-1 cells. We identified 24 sites of phosphorylation, 18 of which are novel sites. This data enhances our knowledge of UBR5 phosphorylation and provides a framework for the study of how phosphorylation affects UBR5 function.     

Pomalidomide restores immune recognition of primary effusion lymphoma through upregulation of ICAM-1 and B7-2

Pomalidomide (Pom) is an immunomodulatory drug that has efficacy against Kaposi’s sarcoma, a tumor caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). Pom also induces direct cytotoxicity in primary effusion lymphoma (PEL), a B-cell malignancy caused by KSHV, in part through downregulation of IRF4, cMyc, and CK1α as a result of its interaction with cereblon, a cellular E3 ubiquitin ligase. Additionally, Pom can reverse KSHV-induced downregulation of MHCI and co-stimulatory immune surface molecules ICAM-1 and B7-2 on PELs. Here, we show for the first time that Pom-induced increases in ICAM-1 and B7-2 on PEL cells lead to an increase in both T-cell activation and NK-mediated cytotoxicity against PEL. The increase in T-cell activation can be prevented by blocking ICAM-1 and/or B7-2 on the PEL cell surface, suggesting that both ICAM-1 and B7-2 are important for T-cell co-stimulation by PELs. To gain mechanistic insights into Pom’s effects on surface markers, we generated Pom-resistant (PomR) PEL cells, which showed about 90% reduction in cereblon protein level and only minimal changes in IRF4 and cMyc upon Pom treatment. Pom no longer upregulated ICAM-1 and B7-2 on the surface of PomR cells, nor did it increase T-cell and NK-cell activation. Cereblon-knockout cells behaved similarly to the pomR cells upon Pom-treatment, suggesting that Pom’s interaction with cereblon is necessary for these effects. Further mechanistic studies revealed PI3K signaling pathway as being important for Pom-induced increases in these molecules. These observations provide a rationale for the study of Pom as therapy in treating PEL and other KSHV-associated tumors.     

The PHD/LAP-domain protein M153R of myxomavirus is a ubiquitin ligase that induces the rapid internalization and lysosomal destruction of CD4

The genomes of several poxviruses contain open reading frames with homology to the K3 and K5 genes of Kaposi's sarcoma-associated herpesvirus (KSHV) and the K3 gene of murine gammaherpesvirus 68, which target major histocompatibility complex class I (MHC-I) as well as costimulatory molecules for proteasomal or lysosomal degradation. The homologous gene product of myxomavirus (MV), M153R, was recently shown to reduce the cell surface expression of MHC-I. In addition, normal MHC-I surface expression was observed in cells infected with MV lacking M153R (J. L. Guerin, J. Gelfi, S. Boullier, M. Delverdier, F. A. Bellanger, S. Bertagnoli, I. Drexler, G. Sutter, and F. Messud-Petit, J. Virol. 76:2912-2923, 2002). Here, we show that M153R also downregulates the T-cell coreceptor CD4 and we study the molecular mechanism by which M153R achieves the downregulation of CD4 and MHC-I. Upon M153R expression, CD4 was rapidly internalized and degraded in lysosomes, whereas deletion of M153R from the genome of MV restored CD4 expression. The downregulation of both CD4 and MHC-I was dependent on the presence of lysine residues in their cytoplasmic tails. Increased ubiquitination of CD4 was observed upon coexpression with M153R in the presence of inhibitors of lysosomal acidification. Surface expression of CD4 was restored upon overexpression of Hrs, a ubiquitin interaction motif-containing protein that sorts ubiquitinated proteins into endosomes. Moreover, the purified PHD/LAP zinc finger of M153R catalyzed the formation of multiubiquitin adducts in vitro. Our data suggest that M153R acts as a membrane-bound ubiquitin ligase that conjugates ubiquitin to the cytoplasmic domain of substrate glycoproteins, with ubiquitin serving as a lysosomal targeting signal. Since a similar mechanism was recently proposed for KSHV K5, it seems that members of the unrelated families of gamma-2 herpesviruses and poxviruses share a common immune evasion mechanism that targets host cell immune receptors.     

The SUMO E3 ligase activity of ORF45 determines KSHV lytic replication

RSK1, an essential cellular kinase for Kaposi’s sarcoma-associated herpesvirus (KSHV) replication, is highly phosphorylated and SUMOylated during KSHV lytic cycle, which determine the substrate phosphorylation and specificity of RSK1, respectively. However, the SUMO E3 ligase responsible for attaching SUMO to RSK1 has not yet been identified. By genome-wide screening, we found that KSHV ORF45 is necessary and sufficient to enhance RSK1 SUMOylation. Mechanistically, KSHV ORF45 binds to SUMOs via two classic SUMO-interacting motifs (SIMs) and functions as a SIM-dependent SUMO E3 ligase for RSK1. Mutations on these ORF45 SIMs resulted in much lower lytic gene expressions, viral DNA replication, and mature progeny virus production. Interestingly, KSHV ORF45 controls RSK1 SUMOylation and phosphorylation via two separated functional regions: SIMs and amino acid 17–90, respectively, which do not affect each other. Similar to KSHV ORF45, ORF45 of Rhesus Macaque Rhadinovirus has only one SIM and also increases RSK1 SUMOylation in a SIM-dependent manner, while other ORF45 homologues do not have this function. Our work characterized ORF45 as a novel virus encoded SUMO E3 ligase, which is required for ORF45-RSK1 axis-mediated KSHV lytic gene expression.     

Ubiquitin enzymes in the regulation of immune responses

Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.     

Kaposi's sarcoma-associated herpesvirus mitochondrial K7 protein targets a cellular calcium-modulating cyclophilin ligand to modulate intracellular calcium concentration and inhibit apoptosis

On viral infection, infected cells can become the target of host immune responses or can go through a programmed cell death process, called apoptosis, as a defense mechanism to limit the ability of the virus to replicate. To prevent this, viruses have evolved elaborate mechanisms to subvert the apoptotic process. Here, we report the identification of a novel antiapoptotic K7 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) which expresses during lytic replication. The KSHV K7 gene encodes a small mitochondrial membrane protein, and its expression efficiently inhibits apoptosis induced by a variety of apoptogenic agents. The yeast two-hybrid screen has demonstrated that K7 targets cellular calcium-modulating cyclophilin ligand (CAML), a protein that regulates the intracellular Ca(2+) concentration. Similar to CAML, K7 expression significantly enhances the kinetics and amplitudes of the increase in intracellular Ca(2+) concentration on apoptotic stimulus. Mutational analysis showed that K7 interaction with CAML is required for its function in the inhibition of apoptosis. This indicates that K7 targets cellular CAML to increase the cytosolic Ca(2+) response, which consequently protects cells from mitochondrial damage and apoptosis. This is a novel viral antiapoptosis strategy where the KSHV mitochondrial K7 protein targets a cellular Ca(2+)-modulating protein to confer resistance to apoptosis, which allows completion of the viral lytic replication and, eventually, maintenance of persistent infection in infected host.     

The Ubiquitin/Proteasome System Mediates Entry and Endosomal Trafficking of Kaposi's Sarcoma-Associated Herpesvirus in Endothelial Cells

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi''s sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process.     

Kaposi’s Sarcoma-Associated Herpesvirus K3 and K5 Proteins Down Regulate Both DC-SIGN and DC-SIGNR

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multicentric Castleman’s disease, primary effusion lymphoma and Kaposi’s sarcoma. In this study, we show that like the C-type lectin DC-SIGN, the closely related DC-SIGNR can also enhance KSHV infection. Following infection, they are both targeted for down modulation and our data indicate that the KSHV MARCH-family ubiquitin ligase K5 is mediating this regulation and subsequent targeting for degradation of DC-SIGN and DC-SIGNR in the context of the virus. The closely related viral K3 protein, is also able to target these lectins in exogenous expressions studies, but only weakly during viral infection. In addition to requiring a functional RING-CH domain, several protein trafficking motifs in the C-terminal region of both K3 and K5 are important in regulation of DC-SIGN and DC-SIGNR. Further exploration of this modulation revealed that DC-SIGN is endocytosed from the cell surface in THP-1 monocytes, but degraded from an internal location with minimal endocytosis in HEK-293 cells. Pull-down data indicate that both K3 and K5 preferentially associate with immature forms of the lectins, mediating their ubiquitylation and degradation. Together, these data emphasize the molecular complexities of K3 and K5, while expanding the repertoire of targets of these two viral proteins.     

Regulated degradation of the HIV-1 Vpu protein through a betaTrCP-independent pathway limits the release of viral particles

Viral protein U (Vpu) of HIV-1 has two known functions in replication of the virus: degradation of its cellular receptor CD4 and enhancement of viral particle release. Vpu binds CD4 and simultaneously recruits the betaTrCP subunit of the SCF(betaTrCP) ubiquitin ligase complex through its constitutively phosphorylated DS52GXXS56 motif. In this process, Vpu was found to escape degradation, while inhibiting the degradation of betaTrCP natural targets such as beta-catenin and IkappaBalpha. We further addressed this Vpu inhibitory function with respect to the degradation of Emi1 and Cdc25A, two betaTrCP substrates involved in cell-cycle progression. In the course of these experiments, we underscored the importance of a novel phosphorylation site in Vpu. We show that, especially in cells arrested in early mitosis, Vpu undergoes phosphorylation of the serine 61 residue, which lies adjacent to the betaTrCP-binding motif. This phosphorylation event triggers Vpu degradation by a betaTrCP-independent process. Mutation of Vpu S61 in the HIV-1 provirus extends the half-life of the protein and significantly increases the release of HIV-1 particles from HeLa cells. However, the S61 determinant of regulated Vpu turnover is highly conserved within HIV-1 isolates. Altogether, our results highlight a mechanism where differential phosphorylation of Vpu determines its fate as an adaptor or as a substrate of distinct ubiquitin ligases. Conservation of the Vpu degradation determinant, despite its negative effect on virion release, argues for a role in overall HIV-1 fitness.     

Cbl-c Ubiquitin Ligase Activity Is Increased via the Interaction of Its RING Finger Domain with a LIM Domain of the Paxillin Homolog,Hic 5

Cbl proteins (Cbl, Cbl-b and Cbl-c) are ubiquitin ligases that are critical regulators of tyrosine kinase signaling. In this study we identify a new Cbl-c interacting protein, Hydrogen peroxide Induced Construct 5 (Hic-5). The two proteins interact through a novel interaction mediated by the RING finger of Cbl-c and the LIM2 domain of Hic-5. Further, this interaction is mediated and dependent on specific zinc coordinating complexes within the RING finger and LIM domain. Binding of Hic-5 to Cbl-c leads to an increase in the ubiquitin ligase activity of Cbl-c once Cbl-c has been activated by Src phosphorylation or through an activating phosphomimetic mutation. In addition, co-transfection of Hic-5 with Cbl-c leads to an increase in Cbl-c mediated ubiquitination of the EGFR. These data suggest that Hic-5 enhances Cbl-c ubiquitin ligase activity once Cbl-c has been phosphorylated and activated. Interactions between heterologous RING fingers have been shown to activate E3s. This is the first demonstration of enhancement of ubiquitin ligase activity of a RING finger ubiquitin ligase by the direct interaction of a LIM zinc coordinating domain.