Polo-like kinase 1 phosphorylation of G2 and S-phase-expressed 1 protein is essential for p53 inactivation during G2 checkpoint recovery

Abstract

In response to G2 DNA damage, the p53 pathway is activated to lead to cell-cycle arrest, but how p53 is eliminated during the subsequent recovery process is poorly understood. It has been established that Polo-like kinase 1 (Plk1) controls G2 DNA-damage recovery. However, whether Plk1 activity contributes to p53 inactivation during this process is unknown. In this study, we show that G2 and S-phase-expressed 1 (GTSE1) protein, a negative regulator of p53, is required for G2 checkpoint recovery and that Plk1 phosphorylation of GTSE1 at Ser 435 promotes its nuclear localization, and thus shuttles p53 out of the nucleus to lead to its degradation during the recovery.

Figure 1

G2 and S-phase-expressed 1 protein interacts with Polo-like kinase 1. (A,B) U2OS cells were transfected with GFP–GTSE1 and transfection-positive cells were selected by G418 (400 μg/ml) for 2 weeks. Cells in different stages of the cell cycle were subjected to immunofluorescence staining with antibodies against (A) Plk1 or (B) γ-tubulin. DNA was stained with DAPI. Scale bars, 10 μm. (C) U2OS cells were treated with nocodazole for 14 h and collected for anti-Plk1, anti-GTSE1 or anti-Erk2 immunoprecipitation (IP), followed by western blot analysis. Erk2 IP was used as a non-specific binding control. (D) U2OS cells were depleted of Plk1 with double-stranded RNA, treated with nocodazole for 14 h and collected for anti-Plk1 IP, followed by western blot analysis. DAPI, 4',6-diamidino-2-phenylindole; Erk2, extracellular signal-related kinase 2; GFP, green fluorescent protein; GTSE1, G2 and S-phase-expressed 1 protein; Plk1, Polo-like kinase 1.

Figure 2

Polo-like kinase 1 phosphorylates G2 and S-phase-expressed 1 protein at Ser 435. (A) Plk1 was incubated with four purified GST–GTSE1 regions (amino acids (aa) 1–170, 171–350, 351–540 and 541–720) in the presence of [γ-³²P]ATP. The reaction mixtures were resolved by SDS–PAGE, stained with Coomassie brilliant blue (CBB), and detected by autoradiography. (B) Plk1 was incubated with the indicated GST–GTSE1 regions as in (A). A longer exposure of ³²P signal is shown in the middle panel. (C) Plk1 was incubated with the indicated forms of GST–GTSE1 fragments (aa 171–260 or 351–440). (D) 293T cells were depleted of Plk1 with dsRNA (lane 3), then transfected with GFP–GTSE1-wt (lanes 1 and 3) or S435A (lane 2), treated with nocodazole, and metabolically labelled with [³²P]orthophosphate. Phosphoproteins were immunoprecipitated with GFP antibodies, resolved by SDS–PAGE and detected by autoradiography. Numbers indicate the relative intensity of ³²P-labelled GTSE1. (E) Plk1 was incubated with GST–GTSE1-aa 351–440 (wt or S435A) in the presence of unlabelled ATP, followed by an anti-pSer 435 western blot. (F) 293T cells were transfected with GFP–GTSE1 in the presence of Plk1 inhibitor (BI 2536) and collected for anti-GFP immunoprecipitation (IP). Then the IP products were incubated with purified Plk1 in the presence of unlabelled ATP, followed by anti-pSer 435 western blot analysis. (G) 293T cells were transfected with GFP–GTSE1 constructs (wt or S435A), treated with nocodazole and immunoblotted. (H) Lysates from mitotic 293T cells transfected with GFP–GTSE1 were treated with λ-phosphatase and immunoblotted. (I) U2OS cells were depleted of Plk1 with dsRNA, treated with nocodazole and immunoblotted. (J) Alignment of GTSE1 protein sequences containing Ser 233 and Ser 435 in different species. dsRNA, double-stranded RNA; GFP, green fluorescent protein; GST, glutathione-S-transferase; GTSE1, G2 and S-phase-expressed 1 protein; Plk1, Polo-like kinase 1; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; wt, wild type.

Figure 3

G2 and S-phase-expressed 1 protein is essential for G2 checkpoint recovery. (A) U2OS cells were transfected with pLKO.1-GTSE1 or pLKO.1 vector for 24 h and immunoblotted. (B) U2OS cells were synchronized with the double thymidine block (16 h treatment with thymidine, 8 h of release and a second 16 h block with thymidine) at the G1/S boundary and GTSE1 was depleted by transfection with pLKO.1-GTSE1 during the 8 h interval and second 16 h blocking period. After release from the double thymidine block for 6 h, the cells were treated with 1.0 μM doxorubicin (Dox) for 1 h, incubated in fresh medium with or without caffeine (Caf) for an additional 6 h. Phospho-histone H3 positivity was determined. (C) Cells as described in (B) were immunoblotted with various antibodies indicated on the left. pT210 is an antibody that recognizes the activated form of Plk1. Numbers at the bottom indicate the relative intensity of p53 compared with actin. (D) On transfection with p53–GFP construct, U2OS cells were subject to synchronization and doxorubicin/caffeine treatment as described in (B). DNA was stained with DAPI. Cells displaying cytoplamsic localization of p53–GFP were quantified in the right panel. Scale bar, 10 μm. (E) U2OS cells were transfected with p53–GFP-wt or p53–GFP-NES (NES-deficient mutant), processed as in (B) and stained with phospho-histone H3. (F) U2OS cells were infected with lentivirus targeting p53, selected by puromycin (10 μg/ml) for 2 weeks, and immunoblotted. (G) The p53-depleted U2OS cells were processed as in (B). (H) H1299 cells were processed as in (B). (I) U2OS cells were depleted of p21 with dsRNA for 24 h, treated with doxorubicin for 1 h and immunoblotted. (J) U2OS cells were depleted of p21 with dsRNA and then processed as in (B). (K) MCF10A cells were processed as in (B). DAPI, 4′,6-diamidino-2-phenylindole; dsRNA, double-stranded RNA; GFP, green fluorescent protein; GTSE1, G2 and S-phase-expressed 1 protein; NES, nuclear export signal; PFT, pifithrin 2.

Figure 4

Phosphorylation of G2 and S-phase-expressed 1 protein by Polo-like kinase 1 is required for G2 checkpoint recovery. (A) U2OS cells were synchronized by the double thymidine block (DTB) protocol, released for 0, 8 and 13 h (left three lanes) or treated with nocodazole (Noc) for 24 h and immunoblotted. (B) U2OS cells were subjected to synchronization/DNA damage as in Fig 3B, released into nocodazole-containing medium with or without caffeine (Caf) and BI 2536 for 6 h and immunoblotted. (C) U2OS cells stably expressing RNAi-resistant GFP–GTSE1 constructs (wt, S435A or S435E) were processed as in Fig 3B with endogenous GTSE1 depleted. Phospho-histone H3 positivity was determined. (D) Samples as in (C) were immunoblotted with antibodies as indicated on the left. (E–G) Plk1 phosphorylation of GTSE1 promotes its nuclear accumulation during G2 checkpoint recovery. (E) U2OS cells transfected with GFP–GTSE1 constructs (wt, S435A or S435E) were subjected to synchronization, DNA damage and caffeine treatment as in Fig 3B and stained with DAPI to determine the subcellular localization of GFP–GTSE1. Scale bar, 10 μm. (F) Quantification of the results in (E). (G) U2OS cells were processed as in (E), but released into medium containing both caffeine and BI 2536. (H–I) Plk1 targets the nuclear localization signal of GTSE1. (H) U2OS cells were transfected with GFP–GTSE1 constructs (wt or R3A) and processed as in (E). Alternatively, GTSE1-expressing cells were incubated with leptomycin B (LMB) for 12 h. Scale bar, 10 μm. (I) Quantification of the results in (H). (J) U2OS cells expressing GFP–GTSE1-R3A were depleted of endogenous GTSE1 and processed as in (C). DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; GTSE1, G2 and S-phase-expressed 1 protein; Plk1, Polo-like kinase 1; R3A, R431A/R432A/R433A; RNAi, RNA interference; wt, wild type.