PIASy controls ubiquitination-dependent proteasomal degradation of Ets-1

Abstract

The ETS transcription factor Ets-1 (E26 transformation-specific-1) plays a critical role in many physiological processes including angiogenesis, haematopoietic development and tumour progression. Its activity can be regulated by post-translational modifications, such as phosphorylation. Recently, we showed that Ets-1 is a target for SUMO (small ubiquitin-like modifier) modification and that PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y], a specific SUMO-E3 ligase for Ets-1, represses Ets-1-dependent transcription. In the present study, we demonstrated that Ets-1 is degraded by the proteasome and that overexpression of PIASy increased the stability of endogenous and ectopically expressed Ets-1 protein by preventing proteasomal degradation. Moreover, knockdown of the endogenous PIASy expression by RNA interference reduced the protein level of endogenous Ets-1. The proteasome inhibitor MG132 reversed this effect. Deletion analysis showed that the TAD (transcriptional activation domain), which has been identified as the interaction domain with PIASy, was also required for Ets-1 ubiquitination and proteasomal degradation. However, the Ets-1 stabilization by PIASy was not due to reduced ubiquitination of Ets-1. Our results suggested that PIASy controls Ets-1 function, at least in part, by inhibiting Ets-1 protein turnover via the ubiquitin-proteasome system.

Figure 1. PIASy overexpression stabilizes Ets-1 against proteasomal degradation

(A) COS-7 cells were transiently transfected with FLAG–Ets-1 (either WT or the 2KR mutant). At 24 h after transfection, cells were treated with vehicle (DMSO) as a control or MG132 (20 μM). After 30 min, CHX (100 μg/ml) was added to the medium. At the indicated time points after the addition of CHX, cell lysates were prepared and FLAG–Ets-1 and β-actin protein levels were detected by immunoblotting with anti-FLAG and β-actin monoclonal antibodies respectively. β-actin was used as a loading control. (B) COS-7 cells were transiently co-transfected with FLAG–Ets-1 (either WT or the 2KR mutant) and Myc–PIASy expression plasmids. After 24 h, CHX (100 μg/ml) was added to the medium. Cell lysates were collected at the indicated time and subjected to immunoblotting with the indicated antibodies, as described for (A). (C) Immunoblot band densities were determined using ImageJ (NIH) and Ets-1 levels were normalized to the loading control, β-actin. The percentage level of Ets-1 was calculated relative to zero time (100%). (D) COS-7 cells were transiently transfected with EGFP (enhanced green fluorescent protein)–PIASy or EGFP control vector. At 24 h after transfection, cells were treated with vehicle (DMSO) as a control, CHX (100 μg/ml) or MG132 (20 μM). After 5 h, cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. (E) COS-7 cells were transiently co-transfected with FLAG–Ets-1 and Myc–PIASy C342A mutant. At 24 h after transfection, cells were treated with CHX (100 μg/ml) or left untreated. After 5 h, cell lysates were prepared and subjected to immunoblotting with the indicated antibodies.

Figure 2. PIASy specifically stabilizes Ets-1

(A) COS-7 cells were transiently co-transfected with FLAG–Ets-1 and Myc–PIASy C342A mutant. At 24 h after transfection, cells were treated with CHX (100 μg/ml) or left untreated. After 5 h, cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. (A) COS-7 cells were transiently co-transfected with FLAG–Ets-1 and EGFP–PIASy or EGFP–PIAS1. CHX treatment and immunoblotting were performed as described for Figure 1(E). (B) Cells were co-transfected and treated as described for (A). Total cellular RNA was extracted and subjected to semi-quantitative RT-PCR. β-Actin was used as a control. (C) COS-7 cells were transiently co-transfected with FLAG–c-Myb or FLAG–AR and Myc–PIASy. CHX treatment and immunoblotting were performed as described for Figure 1(E).

Figure 3. Suppression of PIASy expression down-regulates Ets-1 expression

COS-7 cells were transiently transfected with either control siRNA or PIASy siRNA. At 48 h after transfection, cells were treated with vehicle (DMSO) as a control or MG132 (20 μM). After 5 h, cell lysates were prepared and subjected to immunoblotting with PIASy-, Ets-1- and β-actin-specific antibodies (top panel). Immunoblot band densities were determined using ImageJ (NIH), and PIASy and Ets-1 levels were normalized to the loading control, β-actin (middle and bottom panels respectively). The value for the control siRNA transfected cells in the absence of MG132 was assigned an arbitrary value of 1.0.

Figure 4. Mapping of Ets-1 for the PIASy interaction domain

(A) Schematic diagrams of the Ets-1 constructs used. The two SUMOylation sites (Lys¹⁵ and Lys²²⁷) are indicated. Pointed, Pointed domain; ID, inhibitory domain; ETS, ETS domain. Numbers refer to amino acid residues. The interaction of Ets-1 mutants with PIASy following immunoprecipitation (IP) is summarized. (B) Lysates from COS-7 cells were co-transfected with FLAG–Ets-1 mutants and T7–PIASy were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates were then analysed by immunoblotting with an anti-FLAG antibody (top panel) or an anti-T7 antibody (middle panel). The middle panel shows the Ets-1-bound PIASy. The levels of transfected T7–PIASy protein in the total cell lysates are shown in the bottom panel.

Figure 5. Ets-1 region responsible for stabilization by PIASy

(A) COS-7 cells were transiently co-transected with FLAG–Ets-1 mutants and Myc–PIASy. At 24 h after transfection, CHX (100 μg/ml) was added to the medium and, after 5 h, cell lysates were prepared and subject to immunoblotting with the indicated antibodies. The results presented are representative of three independent experiments. (B) Subcellular localization of Ets-1 mutants. U2OS cells were grown on coverslips and transiently co-transfected with the indicated FLAG–Ets-1 mutants and EGFP–PIASy. At 24 h after transfection, the cells were incubated with an anti-FLAG antibody followed by an Alexa Fluor 568-conjugated secondary antibody, and visualized under a fluorescence microscope. The co-localization of FLAG–Ets-1 and EGFP–PIASy is shown in the merged images.

Figure 6. Ets-1 is ubiquitinated in vivo

(A) COS-7 cells were transiently co-transfected with 3×FLAG–Ets-1 and His₆-ubiquitin as indicated. At 24 h after transfection, the cells were treated with MG132 for 4 h and lysed in buffer containing 6 M guanidinium chloride. His₆-ubiquitinated species were purified with Ni-NTA–agarose beads and analysed by immunoblotting with an anti-FLAG antibody and an anti-ubiquitin antibody. Total cell lysates were subjected to immunoblotting and incubated with an anti-FLAG antibody. (B and C) Experiments analogous to those in (A) were performed using the indicated Ets-1 mutants.

Figure 7. PIASy protects polyubiquitinated Ets-1 proteins from proteasomal proteolysis

COS-7 cells were transiently co-transfected with 3×FLAG–Ets-1, His₆-ubiquitin and Myc–PIASy. Cells were treated with MG132 (20 μM) and CHX (100 μg/ml) for 5 h before lysis. Immunoblotting was performed as described for Figure 6(A).