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. 2010 Apr;38(6):1922-31.
doi: 10.1093/nar/gkp1157. Epub 2009 Dec 21.

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2alpha

Martijn van Hagen  1 , René M Overmeer, Sharareh S Abolvardi, Alfred C O Vertegaal
Affiliations

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2alpha

Martijn van Hagen et al. Nucleic Acids Res. 2010 Apr.

Abstract

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2alpha are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2alpha. Functionally, sumoylation reduced the transcriptional activity of HIF-2alpha. Similar to HIF-1alpha, HIF-2alpha is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2alpha during hypoxia but did not affect the total level of HIF-2alpha. The ubiquitin E3 ligases von Hippel-Lindau and RNF4 control the levels of sumoylated HIF-2alpha, indicating that sumoylated HIF-2alpha is degraded via SUMO-targeted ubiquitin ligases.

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Figures

Figure 1.
Figure 1.
HIF-2α contains two conserved consensus sumoylation sites. (A) Structural domains of HIF-2α. Two consensus sumoylation sites are present in human HIF-2α, LK394EE and LK497IE, which are well conserved in other species. (B) Sequence alignment between HIF-1α and HIF-2α. Both potential HIF-2α sumoylation sites have equivalent sites in HIF-1α, a known SUMO substrate. (C) FLAG-HIF-1α and -2α are conjugated to SUMO-1 and to SUMO-2. HeLa, HeLahis6-SUMO-1 and HeLahis6-SUMO-2 cells were transfected with plasmids encoding FLAG-HIF-1α, -2α or an empty vector control. The cells were grown at 1% O2 for 24 h, lysed and his6-SUMO conjugates were purified. Purified fractions were separated by SDS–PAGE, transferred to membranes and probed with an anti-FLAG antibody. Total cell lysates were included as input controls. Several SUMO-modified forms of FLAG-HIF-1α and -2α could be detected in the his6-SUMO-2 enriched samples whereas only mono-sumoylated forms could be detected in the his6-SUMO-1 enriched samples. bHLH, basic helix-loop-helix; PAS, Per/Arnt/Sim domain; ODD, oxygen dependent degradation domain; N-TAD, N-terminal transactivation domain; C-TAD, C-terminal transactivation domain.
Figure 2.
Figure 2.
K394 in HIF-2α is a SUMO acceptor site. HeLa and HeLahis6-SUMO-2 cells were transfected with plasmids encoding FLAG-HIF-2α wild-type and the indicated mutant proteins. The cells were cultured at 1% O2 for 24 h and lysed 48 h post-transfection. His6-SUMO-2 conjugates were enriched using metal affinity chromatography. Enriched fractions were separated by SDS-PAGE, transferred to membranes and probed with anti-FLAG or anti-SUMO-2/3 antibody. Total cell lysates were included as input controls. K394 was found to be the major SUMO acceptor lysine, whereas K497 appeared not to be used for SUMO conjugation. Total cell lysates were probed with an antibody specific for γ-tubulin as a loading control.
Figure 3.
Figure 3.
SUMO modification of HIF-2α on K394 reduces its transcriptional activity. HeLa cells were cotransfected with wild-type or mutant forms of FLAG-HIF-2α and an HRE-luciferase reporter vector (cartoon). Cells were kept normoxic (21% O2) or cultured hypoxic (1% O2) for 24 h before being lysed in Reporter Lysis Buffer (Promega) for luciferase activity measurements. Control cell lysates were prepared in LDS sample buffer and analysed by immunoblotting to determine the FLAG-HIF-2α expression levels. (A) The transcriptional activity of wild-type FLAG-HIF-2α was compared to the mutants. Mutating either the K394 or E396 in the first consensus sumoylation site resulted in an increase in transcriptional activity over the wild-type both in hypoxic and normoxic samples. Mutations made in the second sumoylation consensus site had no effect on the transcriptional activity of FLAG-HIF-2α. The activity of wild-type HIF-2α under normoxic and under hypoxic conditions was set to 1. A single representative experiment is shown that was carried out in triplicate. The experiment was independently repeated twice with similar results. (B) The expression levels of wild-type and mutant FLAG-HIF-2α proteins were determined by immunoblotting using an anti-FLAG antibody, loading was verified using an anti-γ-tubulin antibody. The increase in transcriptional activity of the sumoylation-impaired HIF-2α mutants was not due to increased expression.
Figure 4.
Figure 4.
Sumoylation does not affect the subcellular localization of FLAG-HIF-2α. HeLa cells were grown on coverslips and transfected with FLAG-HIF-2α expression constructs or empty vector as a control. The cells were incubated at 1% O2 for 24 h before paraformaldehyde fixation. The subcellular localization of wild-type and mutant FLAG-HIF-2α was determined by immunostaining using an anti-FLAG antibody. FLAG-HIF-2α shows a predominant nuclear staining. No difference in localization could be detected between wild-type and mutant proteins. Scale bars are 10 µm.
Figure 5.
Figure 5.
Proteasome inhibition results in an accumulation of SUMO-modified HIF-2α. (A) HeLa cells were cotransfected with plasmids encoding his6-SUMO-2 and FLAG-HIF-2α wild-type and the indicated mutant proteins. The cells were cultured at 1% O2 for 4 h in the presence of 10 µM MG132 or DMSO control and lysed 48 h post-transfection. His6-SUMO-2 conjugates were enriched using metal affinity chromatography. Enriched fractions were separated by SDS-PAGE, transferred to membranes and probed with anti-FLAG or anti-SUMO-2/3 antibody. Total cell lysates were included as input controls. Ponceau S staining was performed to verify equal loading. SUMO-2 conjugates and SUMO-2-modified forms of FLAG-HIF-2α were found to be strongly increased upon proteasome inhibition. Mutation of the major SUMO acceptor site LK394EE reduced HIF-2α-SUMO accumulation whereas mutating the LK497IE consensus site had no effect. Proteasome inhibition had only marginal effects on total FLAG-HIF-2α levels. (B) HeLa cells were transfected with FLAG-HIF-2α wild-type (wt) or the E396A mutant (EA). The cells were cultured at 21% O2 and treated with cycloheximide (CHX) for 3 or 5 h to inhibit protein synthesis. Equal loading was verified by Ponceau S staining. L.C., loading control.
Figure 6.
Figure 6.
Endogenous HIF-2α is conjugated to SUMO-2. HeLaFLAG-SUMO-2 and HeLa cells were cultured for 7 h at 21% (norm.) or 1% O2 (hyp.) in the presence or absence of MG132. FLAG-SUMO-2 conjugates were purified from cell lysates and purified samples were analysed by immunoblotting using an antibody against HIF-2α. Total cell lysates were included as input controls. Sumoylated forms of HIF-2α were specifically detected in the FLAG-SUMO-2 purified fractions from hypoxic cells and accumulated upon proteasome inhibition also in the purified fractions from the normoxic cells. The membrane was reprobed to verify SUMO-2/3 levels in the purified samples. Ponceau S stained protein bands were used as loading controls (L.C.).
Figure 7.
Figure 7.
Sumoylated HIF-2α is regulated by RNF4, SENP1 and VHL. HeLaFLAG-SUMO-2 cells were infected with lentiviruses encoding shRNAs against RNF4, SENP1, RSUME or VHL. The cells were cultured at 1% O2 in the presence of MG132 or DMSO for 7 h and lysed 96 h after infection. FLAG-SUMO-2 conjugates were enriched. Enriched fractions were separated by SDS–PAGE, transferred to membranes and probed with anti-HIF-2α or anti-SUMO-2/3 antibody. Total cell lysates were included as input controls and used to verify the knockdown efficiencies of RNF4 and SENP1. Ponceau S stained proteins were included to show equal loading (L.C.). An increase in the amount of HIF-2α-SUMO-2 conjugates was observed upon knockdown of RNF4, SENP1 and VHL compared to the control sample. RSUME knockdown did not affect the levels of HIF-2α-SUMO-2 conjugates.
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