Plk1-mediated Phosphorylation of Topors Regulates p53 Stability^*

Physical Interaction of Plk1 with Topors

Plk1 expression is elevated in various human tumors including hepatitis B virus-mediated hepatocellular carcinoma (13). The X protein (pX) of hepatitis B virus is implicated in hepatocellular carcinoma development (14). Recent studies have demonstrated the Plk1 is involved in pX-induced hepatocyte transformation using an in vitro pX-expressing cellular model (Studach et al. (28)). Furthermore, Topors was identified as a gene whose depletion via a small interfering RNA library screen rescued pX-expressing cells from DNA damage-induced apoptosis.⁴ Based on these findings, we explored the possibility of potential direct connection between Plk1 and Topors.

To test whether Topors is a binding partner of Plk1, a co-immunoprecipitation experiment was performed. As shown in supplemental Fig. 1A, the Plk1 protein was specifically pulled down by the anti-GFP antibody from lysates of cells expressing GFP-Topors, indicating an association between Topors and Plk1. To examine the interaction between the relevant endogenous proteins, HeLa cells were treated with hydroxyurea or nocodazole, and total cell lysates were subjected to anti-Topors IP followed by anti-Plk1 Western blot. As shown in supplemental Fig. 1B, Plk1 co-immunoprecipitates with Topors using lysates from cells synchronized in S and M phases.

Plk1 Phosphorylates Topors-Ser⁷¹⁸ Both in Vitro and in Vivo

Considering the in vivo interaction between Topors and Plk1, we investigated whether Plk1 phosphorylates Topors. We performed in vitro Plk1 kinase assays, employing affinity-purified GST-Plk1 expressed in baculovirus-infected Hi5 insect cells and affinity-purified GST-Topors, the N-terminal (amino acids 1–510) or C-terminal fragments (aa 511–1045), in the presence of [γ-³²P]ATP. As shown in Fig. 1B, C-terminal Topors, but not N-terminal Topors, was phosphorylated by Plk1 efficiently, indicating that C-terminal Topors is a Plk1 substrate.

To identify the phosphorylation site(s), four shorter Topors fragments (aa 1–250, 251–510, 511–760, and 756–1045) were purified and subjected to Plk1 kinase reactions. Only the fragment containing amino acids 511–760 yielded a strong phosphorylation signal (Fig. 1C). Moreover, a similar experiment with three additional non-overlapping Topors fragments (aa 511–596, 597–678, and 679–760) revealed that that only the fragment spanning amino acids 679–760 was phosphorylated by Plk1 (Fig. 1D). Next, to map the potential phosphorylation site(s) for Plk1, we mutated every serine/threonine in the fragment spanning aa 679–760 into alanine. When compared with the phosphorylation level of WT-Topors (aa 679–760), the phosphorylation level of S718A-Topors was completely abolished, indicating that Ser⁷¹⁸ is the phosphorylation site for Plk1 (Fig. 1E). Significantly, the sequence context of the Plk1 phosphorylation site that we have mapped in Topors is ⁷¹⁷ESSY⁷²⁰. Earlier studies by others have determined that Plk1 prefers a negatively charged residue in the nearby sequence (15–17). Thus, the sequence context of the Plk1 phosphorylation site in Topors (⁷¹⁷ESSY⁷²⁰) matches this rule.

To examine whether Plk1-dependent phosphorylation of Topors occurs in vivo, metabolic labeling experiments were performed. HEK293T cells were transfected with Myc-Topors (WT or S718A), retransfected with double-stranded RNA to deplete Plk1, treated with nocodazole, and metabolically labeled with [³²P]orthophosphate. Topors was immunoprecipitated with an anti-Myc antibody, and the level of Topors phosphorylation was determined (Fig. 1F). The mutation S718A significantly reduced Topors phosphorylation, suggesting that Ser⁷¹⁸ is the major site phosphorylated in vivo under these conditions. Moreover, depletion of Plk1 also reduced the phosphorylation of Topors, indicating that Plk1 does phosphorylate Topors in vivo (Fig. 1F). Moreover, the C-terminal fragment of Topors was expressed and analyzed in vivo. As shown in supplemental Fig. 2, overexpressed GFP-Topors, both in WT and in the S718A mutant, localizes in punctate nuclear regions associated with promyelocytic leukemia nuclear bodies (18). In contrast, GFP-Topors-aa 679–760, both in the WT and in the S718A mutant, is detected throughout the cell with the same localization pattern as GFP (supplemental Fig. 2). Metabolic labeling experiments indicated that this fragment of Topors is not phosphorylated in vivo (supplemental Fig. 2B), likely due to the mislocalization of the protein fragment (supplemental Fig. 2A).

Plk1-dependent Phosphorylation of Topors Negatively Regulates the Protein Level of p53

To explore the role of Plk1-mediated phosphorylation of Topors, U2OS cells were generated that stably express Myc-Topors (WT or S718A). As shown in Fig. 2A, the expression level of Myc-Topors (WT or S718A) is similar in the stably transfected cells.

Because Topors is a dual ubiquitin and SUMO E3 ligase, modulating the stability of p53 (9, 11), we asked whether the Plk1-mediated phosphorylation of Topors regulates p53 stability. To address this question, U2OS cells stably expressing Myc-Topors (WT or S718A) were subjected to an anti-p53 Western blot. Consistent with a previous report (9), expression of WT-Topors led to a slight reduction in the protein level of p53. In striking contrast, the level of p53 protein was significantly elevated in cells expressing S718A-Topors (Fig. 2B, left three lanes). Considering that p53 is stabilized in response to DNA damage, similar experiments were performed in the presence of VP-16, a topoisomerase II inhibitor, used to induce DNA double strand breaks (19). In the presence of VP-16, the p53 level of cells expressing S718A-Topors was still significantly higher than that of cells expressing WT-Topors, suggesting that Plk1 phosphorylation of Topors negatively regulates p53 stability in a DNA damage-independent manner (Fig. 2B, right three lanes). Consistent with the above observation, p21, whose transcription is under control of p53, is clearly elevated in cells expressing S718A-Topors (supplemental Fig. 3A).

The increased level of p53 protein in U2OS cells expressing S718A-Topors could be due to either an increased level of protein translation or a reduced rate of protein degradation. To distinguish between these two possibilities, we quantified the half-life of p53 after treatment with cycloheximide, a protein translation inhibitor (20). First, U2OS cells stably expressing Topors (WT or S781A) were treated with cycloheximide for different times and analyzed by an anti-p53 Western blot. Cycloheximide treatment resulted in a gradual decrease in the level of p53 in cells expressing WT-Topors but not in cells expressing S718A-Topors, suggesting that the S718A mutation prevents p53 degradation (Fig. 2C and supplemental Fig. 4A). This result agrees with our earlier observations that Plk1 depletion leads to an elevation in the level of p53 in HeLa cells (3).

To examine whether this is a general phenomenon or a cell type-dependent event, U2OS cells were depleted of Plk1 by RNA interference (3), treated with cycloheximide, and analyzed by an anti-p53 Western blot. As indicated, the half-life of p53 increased from less than 20 min in control U2OS cells to about 2 h following Plk1 depletion (Fig. 2D and supplemental Fig. 4B). These results indicate that p53 stabilization in response to Plk1 depletion is likely to be a general phenomenon. To further confirm these observations, we analyzed p53 degradation in U2OS cells stably expressing WT-Topors. In these cells, Plk1 depletion also strongly prevented p53 degradation (Fig. 2E and supplemental Fig. 4C) in a pattern almost identical to that in S718A-Topors-expressing cells (Fig. 2C, right four lanes), confirming that Plk1-mediated phosphorylation of Topors promotes p53 degradation. Finally, we compared the stability of p53 in cells expressing different forms of Topors (WT or S718A) after Plk1 depletion. As expected, although p53 degradation was almost completely blocked in S178A-Topors-expressing cells, p53 was also significantly stabilized in cells expressing WT-Topors (Fig. 2F and supplemental Fig. 4D). Because the level of p53 immunostaining in S178A-Topors-expressing cells is very pronounced, only 10% of lysate was loaded in Fig. 2F, right four lanes, resulting in the low level of Mek1 used as our internal control.

To determine the half-life of p53 in cells expressing different forms of Topors (WT or S718A) by quantification, we needed to control p53 Western signals of cells expressing S718A-Topors in a liner range. For that purpose, we deliberately underloaded the S718A samples (Fig. 2F and supplemental Fig. 3B). The amount of lysate loaded in the right four lanes (S718A samples) was only 10% of that in the left four lanes (WT samples), as indicated by Mek1 Western blotting. Therefore, the apparent low Mek1 levels in cells expressing S718A-Topors is not a phenotype (Fig. 2F and supplemental Fig. 3B) because the Mek1 levels of cells expressing S718A-Topors were the same as those of cells expressing WT-Topors if the same amount of lysates was loaded (Fig. 2C).

Plk1-mediated Phosphorylation of Topors Modulates Sumoylation Versus Ubiquitination of p53

Considering that Topors functions both as a ubiquitin and as a SUMO-1 E3 ligase for p53, we asked whether the Plk1-dependent phosphorylation of Topors is involved in the sumoylation or ubiquitination of p53. Toward this end, U2OS cells stably expressing Topors (WT or S718A) were transfected with YFP-SUMO-1 and analyzed by an anti-p53 Western blot. As indicated in Fig. 3A, multiple SUMO-1-modified forms of p53 were detected in lysates from cells expressing S718A-Topors. By contrast, sumoylated forms of p53 were absent in WT-Topors-expressing or non-transfected U2OS cells, suggesting that Plk1-mediated phosphorylation of Topors inhibits its sumoylation activity toward p53. This is not due to a loading difference because sumoylated forms of p53 can be easily detected even with only 5% of the lysate from S718A-Topors-expressing cells (supplemental Fig. 5A). The increased level of p53 sumoylation in cells expressing S718A-Topors was also confirmed by anti-SUMO-1 Western blotting (supplemental Fig. 5B). To provide more evidence that Plk1 activity regulates sumoylation of p53, U2OS cells stably expressing Topors (WT or S718A) were co-transfected with YFP-SUMO-1 and pBS/U6-Plk1, the vector used to deplete Plk1 previously (3). When compared with cells with a normal Plk1 level, both untransfected cells and WT-Topors-expressing cells accumulated sumoylated forms of p53 upon Plk1 depletion (Fig. 3B). Based on these observations, we conclude that Plk1-mediated phosphorylation of Topors at Ser⁷¹⁸ negatively regulates sumoylation of p53 in mammalian cells.

In addition, we examined whether Plk1 phosphorylation of Topors also regulates the ubiquitination of p53. Accordingly, U2OS cells stably expressing Topors (WT or S718A) were transfected with His-ubiquitin and subjected to an anti-p53 IP followed by an anti-p53 Western blot. As shown in Fig. 3C and supplemental Fig. 5C, strong ubiquitin-modified forms of p53 were observed in both control and WT-Topors-expressing cells, whereas ubiquitinated forms p53 were absent in S718A-Topors-expressing cells. These results demonstrate that phosphorylation of Topors at Ser⁷¹⁸ is required for ubiquitination of p53. Taken together, our data strongly support the notion that Plk1-mediated phosphorylation of Topors suppresses the sumoylation of p53, inducing instead p53 ubiquitination, which results in p53 degradation.