Hypoxia Inactivates the VHL Tumor Suppressor through PIASy-Mediated SUMO Modification

The hypoxic microenvironment contributes to embryonic development and tumor progression through stabilization of the potent transcriptional factor HIFα. In normoxia, the tumor suppressor protein VHL acts as an E3 ubiquitin ligase to target HIFα for proteolytic destruction. Increasing evidence shows that VHL is a multifunctional adaptor involved in inhibition of HIFα-dependent and independent cellular processes. However, the molecular effect of hypoxic stress on VHL functions remains elusive. Here we report that PIASy, a SUMO E3 ligase upregulated in hypoxia, interacts with VHL and induces VHL SUMOylation on lysine residue 171. Moreover, PIASy-mediated SUMO1 modification induces VHL oligomerization and abrogates its inhibitory function on tumor cell growth, migration and clonogenicity. Knockdown of PIASy by small interfering RNA leads to reduction of VHL oligomerization and increases HIF1α degradation. These findings reveal a unique molecular strategy for inactivation of VHL under hypoxic stress

EC(5)S Ubiquitin Complex Is Recruited by KSHV Latent Antigen LANA for Degradation of the VHL and p53 Tumor Suppressors

Cellular protein degradation pathways can be utilized by viruses to establish an environment that favors their propagation. Here we report that the Kaposi's sarcoma–associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) directly functions as a component of the EC(5)S ubiquitin complex targeting the tumor suppressors von Hippel-Lindau (VHL) and p53 for degradation. We have characterized a suppressor of cytokine signaling box-like motif within LANA composed of an Elongin B and C box and a Cullin box, which is spatially located at its amino and carboxyl termini. This motif is necessary for LANA interaction with the Cul5–Elongin BC complex, to promote polyubiquitylation of cellular substrates VHL and p53 in vitro via its amino- and carboxyl-terminal binding domain, respectively. In transfected cells as well as KSHV-infected B lymphoma cells, LANA expression stimulates degradation of VHL and p53. Additionally, specific RNA interference–mediated LANA knockdown stabilized VHL and p53 in primary effusion lymphoma cells. Thus, manipulation of tumor suppressors by LANA potentially provides a favorable environment for progression of KSHV-infected tumor cells

Kaposi's Sarcoma Herpesvirus Upregulates Aurora A Expression to Promote p53 Phosphorylation and Ubiquitylation

Aberrant expression of Aurora A kinase has been frequently implicated in many cancers and contributes to chromosome instability and phosphorylation-mediated ubiquitylation and degradation of p53 for tumorigenesis. Previous studies showed that p53 is degraded by Kaposi's sarcoma herpesvirus (KSHV) encoded latency-associated nuclear antigen (LANA) through its SOCS-box (suppressor of cytokine signaling, LANASOCS) motif-mediated recruitment of the EC5S ubiquitin complex. Here we demonstrate that Aurora A transcriptional expression is upregulated by LANA and markedly elevated in both Kaposi's sarcoma tissue and human primary cells infected with KSHV. Moreover, reintroduction of Aurora A dramatically enhances the binding affinity of p53 with LANA and LANASOCS-mediated ubiquitylation of p53 which requires phosphorylation on Ser215 and Ser315. Small hairpin RNA or a dominant negative mutant of Aurora A kinase efficiently disrupts LANA-induced p53 ubiquitylation and degradation, and leads to induction of p53 transcriptional and apoptotic activities. These studies provide new insights into the mechanisms by which LANA can upregulate expression of a cellular oncogene and simultaneously destabilize the activities of the p53 tumor suppressor in KSHV-associated human cancers

Distinct Domains of LANA Interact with Tumor Suppressors <i>p53</i> and <i>VHL</i>

<div><p>(A) The amino-terminal domain of LANA binding <i>VHL</i> and carboxyl-terminal domain binding <i>p53</i> in vitro. The cDNA encoding N (1–340 aa), C (762-1162 aa), and NC (1–327^929-1162 aa) LANA with myc tag, HA-VHL, and <i>p53</i> were translated in a coupled transcription/translation system in the presence of [³⁵S]methionine. Fifteen-microliter aliquots of the translation products were immunoprecipitated with anti-myc antibodies. Immunoprecipitated proteins (IP) and a 1-μl aliquot of the transcription/translation reaction (Input) were fractionated by SDS-PAGE and detected by autoradiography. RD, relative density.</p><p>(B) LANA is associated with <i>p53</i> and <i>VHL</i> in cells. Saos-2 cells were cotransfected with expression vector encoding the indicated proteins. The lysates underwent IP using anti-myc antibodies. Crude extracts (left panels) and immune complexes (right panels) were separated by SDS-PAGE and IB with antibodies indicated. Asterisk indicates IgG heavy chain.</p></div

Binding to the Elongin BC Complex Stabilizes LANA

<div><p>(A) The stabilities of WT LANA and its SOCS motif mutant. Five million Saos-2 cells were transfected with 5 μg of LANA-WT (left panel) or LANA-ΔSOCS (right panel) expression vector in the presence of 5 μg of Elongin B and 5 μg of Elongin C expression vectors, as indicated. Twenty-four hours after transfection, cells were treated with 100 μg/ml cyclohexamide (CHX) for different times (0, 5, 10, and 24 h). Aliquots (40 μg) of each whole-cell extract underwent IB with the indicated antibodies.</p><p>(B) The relative levels of WT LANA and its SOCS mutant coexpression with Elongin BC complex. Protein density was quantitated by densitometry of immunoblots using Odyssey Image v1.2 from three separate experiments.</p></div

The LANA–Elongin BC Complex Assemble with the Cul5/Rbx1 Module

<p>(A) LANA protein associates with the Cul5/Rbx1 module but not Cul2/Rbx1 module, and (B) Elongin BC complex increases interaction of LANA with the Cul5/Roc1 module. Saos-2 cells were transfected with expression vector encoding the indicated proteins in the figure. The lysates underwent IP using anti-myc antibodies. Crude extracts (left panels) and immune complexes (right panels) were separated by SDS-PAGE and IB with the indicated antibodies.</p

LANA contains a unique SUMO2-interacting motif.

<p>(<b>A</b>) LANA interacts with SUMO-2 and is sensitive to hypoxia. HEK293 cells were co-transfected with expression plasmids as indicated in the figure, and individually treated with or without hypoxia (100 µM CoCl₂ or 1% O₂) at 24 hr post-transfection for overnight before harvest. Cell extracts were subjected to co-immunoprecipitated (co-IP) and immunoblotting (IB) as indicated. The same membrane was stripped and reblotted (reIB) with indicated antibodies. The relative density (RD) of LANA-interacting SUMO-1 or SUMO-2 modified substrates [(SUMO1/2)n-sb] is shown. The position (>170 kDa) of hypoxia-sensitive SUMO-2 modified substrate [(SUMO2)n-sb] from whole cell lysate (WCL) is enlarged. The reduced level of (SUMO2)n-sb is highlighted as “high” and “low”. (<b>B</b>) Schematic representation of different LANA truncated mutants and the affinity of their interaction with SUMO-1/2. The relative affinity of LANA with SUMO-1 or SUMO-2 was summarized from <i>supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003750#ppat.1003750.s001" target="_blank">Figure S1</a></i> and <i>panel C</i>. The homology of SUMO-interacting motifs of LANA (LANA^SIM: SIM1 and SIM2) with the cellular conserved SIM (motifs 1, 2 and 3) sequences, and residue positions of each LANA mutant is indicated. Su2, SUMO-2; SIM, SUMO-interacting motif; AD, acidic domain. (<b>C</b>) An intact SIM motif is required for LANA to interact with SUMO-2. HEK293 cells were co-transfected with expression plasmids as indicated. At 48 hr post-transfection, whole cell lysates (WCL) were subjected to co-immunoprecipitated (co-IP) and immunoblotting (IB) as indicated. The same membrane was stripped and reblotted (reIB) with indicated antibodies. (<b>D</b>) The SIM motif of LANA is required for interacting with SUMO-2 not SUMO-1 <i>in vitro</i>. <i>In vitro</i>-translated, radiolabeled wild type (WT) LANA and its mutants (ΔAD, ΔAD,SIM) was individually incubated with His-SUMO-1 or His-SUMO-2 recombinant proteins with Nickel-agarose (Ni-NTA) beads. His-SUMO-1 and His-SUMO-2 recombinant proteins are shown by Coomassie staining at the bottom panel. Bound complexes were analyzed by autoradiography. Luciferase (Luc) was used as negative control. Input was used at 5%.</p

The LANA^SIM deletion reduces the co-localization of LANA with SUMO-2 on chromatin DNA.

<p>HEK293 cells co-transfected GFP-SUMO-2 with vector (<b>A</b>) alone, wild type (WT) or SIM-deleted (ΔSIM) mutant of LANA NC with myc tag (<b>B</b>) was individually cultured on coverslips, fixed with 3% paraformaldehyde, and then stained with anti-myc antibody as indicated. Bottom panels show the magnified view and arrowheads point examples of LANA and SUMO-2 co-localization on chromosome DNA within the outline region. Nuclei were counterstained with DAPI. Scale bars, 2 µm. Right panel shows schematic of co-localization (yield white) of LANA (red) and SUMO-2 (green) with host chromatin DNA (blue) and the percentage of co-localization from 50 counted cells. (<b>C</b>) Sequential ChIP was performed first with anti-GFP antibody or mouse IgG followed by a second ChIP with anti-myc antibody. Quantitative PCR was performed using TR specific primer. (<b>D</b>) Hypoxia reduces the co-localization of LANA with SUMO-2. BC3 cells were subjected to hypoxia (1% oxygen) treatment for overnight. Endogenous LANA and SUMO-2 were individually stained by LNA1 (rat) and SUMO-2 (rabbit) antibodies. SUMO-2 non-colocalization with LANA was highlighted by dot box. Percentage of co-localization from 10 counted cells.</p

LANA Promotes Polyubiquitylation of Tumor Suppressors <i>p53</i> and <i>VHL</i>

<div><p>(A) LANA induces <i>p53</i> and <i>VHL</i> polyubiquitylation. Saos-2 (left panel) or 786-O (right panel) cells were cotransfected with different combinations of myc-LANA (WT and ΔSOCS), myc-<i>p53</i> (or HA-VHL), and HA-Ub as indicated. At 48 h posttransfection, transfected cells were harvested, lysed, and protein normalized. Total protein (40 μg) was subjected to resolve and IB assays against myc (LANA), <i>p53</i> (or <i>VHL</i>), and β-actin. The data showed LANA can induce <i>p53</i> and <i>VHL</i> ubiquitylation in vivo.</p><p>(B) The LANA–Elongin BC–Cul5–Rbx1 complex induces <i>p53</i> and <i>VHL</i> polyubiquitylation in vitro. The cell lysates of WT LANA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020116#ppat-0020116-g003" target="_blank">Figure 3</a>C, lane 4) or SOCS-box–like motif deletion mutant (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020116#ppat-0020116-g003" target="_blank">Figure 3</a>C, lane 3) as control was subjected to anti-myc immunoaffinity purification. The purified LANA immune complex (IC) was incubated with various combinations of Uba1 (E1), Ubc5a (E2), His-Ub, or GST-VHL (or GST-p53) in the presence of ATP in vitro. The proteins were separated by SDS-PAGE and IB against GST antibody. Left panel, GST-p53; right panel, GST-VHL.</p></div