Plk1 phosphorylates Topors at Ser⁷¹⁸in vitro and in vivo. A, schematic representation of the domain structure of Topors. Two separate regions encoding putative p53-binding domains are aa 456–731 and 854–916. Amino acid residues in the putative Ring finger motif are shown in a black box. PEST, sequences rich in Pro, Glu, Ser, and Thr; RS domain, Arg- and Ser-rich domain; NLS, nuclear localization sequence; NB, nuclear bodies. B, purified Plk1 was incubated with purified GST-Topors (aa 1–510) or GST-Topors (aa 511–1045) for 30 min at 30 °C in the presence of [γ-³²P]ATP (³²P). Reaction mixtures were resolved by SDS-PAGE followed by autoradiography. Coom., Coomassie Blue. C and D, Plk1 phosphorylates Topors (aa 679–760). Purified Plk1 was incubated with purified GST-Topors fragments (aa 1–250, 251–510, 511–760, 756–1045, 511–596, 597–678, and 679–760). Kinase assays were performed as described in B. E, Ser⁷¹⁸ of Topors is a Plk1 phosphorylation site in vitro. Purified Plk1 was incubated with the indicated serine to alanine Topors (aa 679–760) mutants and analyzed as in B. F, Topors is phosphorylated in vivo at Ser⁷¹⁸ by Plk1. HEK293T cells were transfected with WT-Topors-Myc (lanes 1 and 3) or S718A-Topors-Myc (lane 2) and depleted of Plk1 by using double-stranded RNA targeting Plk1 (lane 3). After overnight incubation, cells were treated with nocodazole for 10 h and metabolically labeled with [³²P]orthophosphate. Phosphoproteins were immunoprecipitated with anti-Myc antibodies, resolved by SDS-PAGE, and subjected to autoradiography. Relative ³²P (Rel. ³²P) incorporations of Topors are indicated on the bottom.

The protein level of p53 is negatively regulated by Plk1-dependent phosphorylation of Topors. A, Western blots of extracts of control U2OS cells or U2OS cells stably expressing Myc-Topors (WT or S718A) using Myc (top) or Topors (bottom) antibodies. Endo., endogenous. B, U2OS cells stably expressing Myc-Topors (WT or S718A) were treated with or without 200 μm VP-16 for 18 h and analyzed by anti-p53 immunoblot. C, U2OS cells stably expressing Myc-Topors (WT or S718A) were treated with 75 μg ml⁻¹ of cycloheximide (CHX) for the indicated time course and analyzed as in B. The ratios between p53 and Mek1 are indicated on the bottom. D, U2OS cells were transfected with pBS/U6-Plk1 to deplete Plk1, treated with cycloheximide as indicated, and harvested for anti-p53 immunoblot. For control samples, cells were transfected with pBS/U6 empty vector only. RNAi, RNA interference. E, U2OS cells stably expressing WT-Topors-Myc were depleted of Plk1 as in D, incubated overnight, and treated with cycloheximide for the indicated times. F, U2OS cells stably expressing Myc-Topors (WT or S718A) were depleted of Plk1, incubated overnight, and treated with cycloheximide for the indicated times. The amount of lysate loaded in the right four lanes was 10% of that in the left four lanes, as indicated by Mek1 Western blotting.

Plk1-mediated phosphorylation of Topors suppresses sumoylation but increases ubiquitination of p53. A, U2OS cells stably expressing Myc-Topors (WT or S718A) were transfected with YFP-SUMO-1, incubated for 1 day, and analyzed by anti-p53 Western blotting. B, U2OS cells stably expressing Myc-Topors (WT or S718A) were co-transfected with YFP-SUMO-1 and pBS/U6-Plk1 at a ratio of 4:3, incubated for 1 day, and analyzed as in A. For cells expressing S718A-Topors (lanes 3 and 6), only 10% of lysate was loaded when compared with the rest of samples. C, U2OS or U2OS cells expressing Myc-Topors (WT or S718A) were transfected with His-ubiquitin, incubated for 1 day, and harvested. Lysates were subjected to anti-p53 IP followed by anti-p53 Western blotting.