Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity

Abstract

The activity of the p53 tumor suppressor protein and the c-Jun protooncogene is regulated by posttranslational modifications, such as phosphorylation or ubiquitination. In addition, covalent attachment of the ubiquitin-like modifier SUMO appears to modulate their transcriptional activity. Sumoylation proceeds via an enzymatic pathway that is mechanistically analogous to ubiquitination, but requires a different E1-activating enzyme and Ubc9, a SUMO-specific E2-conjugating enzyme. Here, we show that two members of the PIAS family, PIAS1 and PIASxbeta, act as specific E3-like ligases that promote sumoylation of p53 and c-Jun in vitro and in vivo. The PIAS proteins physically interact with both p53 and c-Jun. In addition, they bind to Ubc9, suggesting that they recruit the E2 enzyme to their respective substrate. The SUMO ligase activity requires the conserved zinc-finger domain, which is distantly related to the essential RING-finger motif, found in a subset of ubiquitin ligases. Furthermore, similar to RING-type ubiquitin ligases, PIASxbeta can catalyze its own modification. Hence, these data further extend the analogy between the ubiquitin and SUMO pathway. Strikingly, PIAS proteins strongly repress the transcriptional activity of p53, suggesting that the PIAS-SUMO pathway plays a crucial role in the regulation of p53 and presumably other transcription factors.

Figure 1

PIAS 1 and PIASxβ promote sumoylation of p53 in vitro. The indicated proteins were in vitro translated and incubated either in the absence (−) or presence (+) of the assay mix containing recombinant E1 (Aos1/Uba2), Ubc9, and SUMO-1. Where indicated, GST-PIAS1 (A), GST-PIASxβ (B and C), or GST-PIASxβ^C362S (D) was added at concentrations of 10, 50, and 100 ng.

Figure 2

PIAS induces sumoylation of recombinant p53. p53 expressed as a GST-fusion protein in E. coli was used for in vitro sumoylation as described in Fig. 1. GST-p53 was used at a concentration of 50 ng and GST-PIASxβ at 250 ng. p53 was detected by immunoblotting by using an anti-p53 antibody.

Figure 3

PIAS 1 and PIASxβ promote sumoylation of c-Jun in vitro. The indicated proteins were in vitro translated and incubated either in the absence (−) or presence (+) of the assay mix as described in Fig. 1. In A, GST-PIASxβ was added at a concentration of 100 ng, where indicated. In B, GST-PIASxβ and GST-PIAS1 were used at concentrations of 50 and 100 ng.

Figure 4

p53 and c-Jun bind to PIAS1 and PIASxβ. ³⁵S-labeled in vitro translated p53 (A), c-Jun (B), and luciferase (C), serving as a negative control, were incubated with the indicated GST fusion proteins bound to glutathione-Sepharose beads. Beads were washed, eluted, and subjected to electrophoresis and autoradiography to reveal the bound radiolabeled proteins. The input representing 20% for each radiolabeled protein is shown.

Figure 5

PIASxβ binds to Ubc9. ³⁵S-labeled in vitro translated PIASxβ (A) or luciferase (B), serving as a negative control, was incubated with the indicated GST fusion proteins as in Fig. 4.

Figure 6

PIASxβ catalyzes its own modification.³⁵S-labeled in vitro translated PIASxβ or PIASxβ^C362S was incubated either in the absence (−) or presence (+) of the assay mix containing recombinant E1 (Aos1/Uba2), Ubc9, and SUMO-1. GST-PIASxβ (100 ng) was added where indicated.

Figure 7

PIAS 1 and PIASxβ enhance sumoylation of p53 in vivo. HeLa cells were transfected with p53 alone or together with SUMO-1 (SUMOGG) as indicated. Plasmids expressing a Flag-tagged PIAS1 (A), an SV5-tagged PIASxβ (B), and the SV5-tagged PIASxβ^C362S mutant (C) were cotransfected where indicated. Cells were directly lysed in SDS-sample buffer, and samples were analyzed by Western blotting with antibodies as indicated. All samples had been cotransfected with plasmids expressing green fluorescent protein to verify equal transfection efficiency by immunoblotting with an anti-GFP antibody.

Figure 8

PIAS down-regulates the transcriptional activity of p53. HeLa cells were transiently transfected with the pRGC luciferase reporter plasmid that harbors a p53 DNA binding site in its promoter, together with the plasmids as indicated. Values represent the average of four independent experiments with triplicate transfections after normalization for the internal control renilla luciferase activity. Activities are expressed relative to p53-induced promoter activity, which was set at 100.