BC-box protein domain-related mechanism for VHL protein degradation

Abstract

The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.

Keywords: hypoxia; oncoviral proteins; ubiquitylation.

Fig. 1.

Gam1 WT decreases cellular levels of VHL, stabilizes HIF 1α, and increases the transactivational function of HIF 1α. (A) Immunoblotting (IB) showing Glut1 and HIF1α stabilization. HeLa Tet-on cells were induced (DOX) to express Myc Gam1 WT. Lysates were made in SDS lysis buffer at the indicated times and probed as indicated. Loading controls: Vinculin and Tubulin. (B) Luciferase assay. HeLa cells transfected with HRE (Hypoxia Responsive Element)-Luciferase reporter plasmid together with either empty vector or myc Gam1 wild-type (WT). Results are reported as fold induction of luciferase activity in relative light units per second (RLU/s) normalized to the control (empty vector) where n = 3 ± SD. (C) Quantitative RT-PCR. Results are reported as fold induction of CAIX mRNA relative to the control (empty vector) and normalized respectively to GADPH mRNA (to which the arbitrary value of 1 was assigned) where n = 3 ± SD. (D) IB of VHL and HIF 1α in HeLa cells transiently transfected with Myc Gam1 WT and Myc Gam1 LL/AA. Loading control: Tubulin and Vinculin.

Fig. 2.

Gam1 WT and not Gam1 LL/AA cause ubiquitylation and proteasome-dependent decrease of VHL. (A) Tet-on HEK293T cells were induced (DOX). One hour after induction, MG132 or DMSO was added to the medium. nUb was used as a control for proteasome inhibition. Loading control: Tubulin. (B) Phoenix cells were transfected with HA VHL, Flag Ub, and myc Gam1 (WT or mutant LL/AA), harvested, and lysed in denaturing SDS-lysis buffer. One milligram of crude extract was immunoprecipitated (IP) against HA tag and analyzed by IB. Anti-Flag antibody detected ubiquitinated HA VHL. Loading control: Tubulin.

Fig. 3.

SOCS domain BC-box mutant does not decrease VHL protein levels. (A) U2OS cells were transfected with Flag E4orf6, Flag SOCS1, or Flag SOCS3. Lysates were analyzed as depicted. Loading control: Tubulin and Vinculin. (B) Schematic representation of the EGFP-tagged SOCS domain, subcloned from full-length SOCS1 and SOCS3. Box mutants were constructed by specific mutations (*) within the BC box. (C) HeLa cells transfected with SOCS box, and their respective mutants were harvested and IP for GFP. IB for Elongin C shows mutant proteins unable to bind Elongin C. (D) Protein expression of SOCS box and their respective mutants in HeLa cells. Lysates were IB for VHL. Loading control: Actin.

Fig. 4.

Cotransfection of EloB/C does not affect VHL protein decrease. (A) HeLa cells were cotransfected with a panel of SOCS domain proteins and their respective BC-box mutant proteins. EloB/C were cotransfected as indicated (+ or −). Lysates were probed against VHL, EloB/C, and SAE1. Loading control: Actin. Decrease in SAE1 is specific to Gam1 WT protein expression and is the positive control. (B) Graph of VHL protein levels (from immunoblot in A) based on pixel-intensity values measured from plot profiles of individual lanes using ImageJ. (C–E) Immunoblots of transfected proteins with loading controls.

Fig. 5.

Cullin2 and Cullin5 are involved in VHL decrease. (A) HeLa cells were transfected with shCul2 and shCul5 or control vector. VHL levels relative to Actin loading show a less significant decrease in VHL upon Cul2 and Cul5 knockdown in SOCS domain-transfected cells. (B) Transient expression of the transfected proteins is shown by IB. (C) HeLa cells selected as described in A were transfected as depicted. Flag Ub was IP under SDS lysis conditions (SI Materials and Methods). Loading control: Tubulin.

Fig. 6.

Heat shock and viral infection decreases VHL protein. (A) Tet-on inducible HeLa cells were subjected to heat-shock treatment (HS) or kept at 37 °C as control. VHL protein decrease upon heat shock, as shown in the representative IB, is significant and consistent (P < 0.05, n = 3). Control: Hsp70 asterisk indicates the stress-induced band. A graphical representation of SOCS1 mRNA in control and HS cells shows a statistically significant (P < 0.006, n = 3) and reproducible increase of SOCS1 mRNA. (B) HeLa cells were either mock infected or infected with Ad5ΔE3. Twenty-four hours after infection, lysates were probed for VHL and Glut1. Infection was confirmed by probing with DBP. Loading control: Tubulin. (C) Primary Human Keratinocytes expressing viral proteins (SI Materials and Methods). Lysates were probed for p53 and VHL protein. Loading control: GAPDH.

Fig. 7.

Model showing VHL protein decrease upon expression of SOCS domain-containing proteins of viral and cellular origin. Infection by viral proteins containing the SOCS domain or increased protein levels of cytokine signaling mediators SOCS1 and SOCS3 decrease VHL levels. BC-box mutants in the SOCS domain fail to assemble a functional Cullin2 and Cullin5 ligase complex, resulting in reduced VHL protein ubiquitination and its subsequent stabilization. VHL protein decrease might also be mediated by other unknown ligases as in the case of a heat-shock response or in cellular states when alternate pathways are activated. The consequent stabilization and activation of HIF 1 at the transcriptional level might provide a favorable environment for cell survival even under conditions of cellular stress.