Polo-like kinase 1 phosphorylation of p150Glued facilitates nuclear envelope breakdown during prophase

Abstract

Nuclear envelope breakdown (NEBD) is an essential step during the G2/M transition in higher eukaryotic cells. Increasing evidence supports the notion that both microtubules and microtubule-associated motor proteins are critical regulators of NEBD. Although it has been described that p150(Glued), the major component of the dynein/dynactin complex, localizes in the nuclear envelope during prophase, the exact role of p150(Glued) and its regulation during NEBD are largely elusive. Polo-like kinase 1 (Plk1), the best characterized Ser/Thr kinase, is involved in mitotic entry in several systems; however, the targets of Plk1 during NEBD are unknown. Herein, we show that in mammalian cells both Plk1 and p150(Glued) regulate NEBD and that Plk1 interacts with and phosphoryates p150(Glued) during NEBD at prophase. Using various approaches, we showed that Plk1 phosphorylates p150(Glued) at Ser-179 and that the pS179 epitope is generated at the nuclear envelope of prophase cells. Significantly, Plk1-mediated phosphorylation of p150(Glued) at Ser-179 positively regulates its accumulation at the nuclear envelope during prophase. Finally, we found that cells expressing the Plk1-unphosphorylatable mutant (p150(Glued)-S179A) arrest at G2, as indicated by reduced NEBD, increased levels of cyclin B and phospho-H3, but a decreased level of Cdc2 kinase activity. Taking these data together, we conclude that Plk1 phosphorylation of p150(Glued) might be one major pathway of NEBD regulation.

Fig. 1.

The p150^Glued is a Plk1 substrate in vitro and in vivo. (A) GST-Plk1 was incubated with four purified GST-p150^Glued fragments (amino acids 1–310, 309–620, 618–964, 963–1282) in the presence of [γ-³²P]ATP. The reaction mixtures were resolved by SDS/PAGE, stained with Coomassie Brilliant Blue (Coom.), and detected by autoradiography. (B) Plk1 was incubated with indicated forms of GST-p150^Glued-N (amino acids 1–310) as in A. (C) Purified Plk1 was incubated with purified GST- p150^Glued-N (WT or S179A) in the presence of unlabeled ATP, followed by an anti-pS179 Western blot. (D) HEK293T cells were transfected with GFP-p150^Glued constructs (WT or S179A), treated with or without nocodazole, and immunoblotted with antibodies, indicated on the left. (E) Mitotic lysates from 293T cells expressing GFP-p150^Glued were treated with λ-phosphatase, followed by Western blotting. (F) HeLa cells were cotransfected with pBS/U6-Plk1 and pBabe-puro at a ratio of 7:1. At 1 d posttransfection, cells were selected with puromycin for additional 36 h. After floating cells were washed away, attached cells were treated with nocodazole for 12 h and immunoblotted.

Fig. 2.

Temporal and spatial regulation of the pS179-p150^Glued epitope in HeLa cells. (A) Cells were treated with nocodazole for 16 h (lanes 2–7), collected by mitotic shake-off, released for different times, and immunoblotted. In lane 1, cells were treated with thymidine for 24 h to block at G1. (B) Cells at indicated phases of cell cycle were costained with antibodies against pS179 and p150^Glued. DNA was stained with DAPI. (C and D) Cells were transfected with siRNA to deplete p150^Glued (C) or treated with BI2536 to inhibit Plk1 activity (D), and costained with antibodies against p150^Glued and pS179. Mitotic cells are indicated by the arrows. (E) A series of Z-scans of a prophase cell stained with p-S179 and lamin A/C antibodies. (Scale bars, 5 μm.)

Fig. 3.

Both Plk1 and p150^Glued are involved in NEBD during prophase in HeLa cells. (A and B) Cells were synchronized with a double thymidine block (DTB, thymidine block for 16 h, release for 8 h, and a second thymidine block for 16 h) at the G1/S boundary with Plk1 being depleted during the 8-h interval. Upon release for 11 h, the cells were stained with lamin A/C antibodies. (A) Representative images of Plk1-depleted cells in G2, prophase, and prometaphase. (B) Plk1 depletion retards NEBD in prophase. (C) Plk1 inhibition prevents the formation of NE invaginations in prophase cells. Cells were synchronized with the DTB, released for 11 h in the presence of BI2536, and stained as in A. Prophase cells with clear NE invaginations were quantified. (D) Overexpression of p150^Glued led to a premature NEBD. Cells expressing GFP-p150^Glued were processed and stained as in A. (Scale bars, 5 μm.)

Fig. 4.

Plk1-mediated phosphorylation of p150^Glued is essential for its NE accumulation in prophase. (A) p150^Glued localizes to the NE in prophase, but not in interphase. Randomly growing U2OS cells were costained with antibodies against p150^Glued and lamin A/C. (B and C) Accumulation of p150^Glued at the NE in prophase is Plk1-dependent. (B) U2OS cells growing on coverslips were transfected with siRNA to deplete Plk1, blocked with thymidine for 20 h, released for 11 h, and immunostained with indicated antibodies. (C) Quantification of the results of NE accumulation of p150^Glued. (D and E) Localization of p150^Glued at the NE was reduced by the S179A mutation. (D) U2OS cells stably expressing RNAi-resistant GFP-p150^Glued (WT or S179A) were transfected with siRNA to deplete endogenous p150^Glued, synchronized, and processed as in B. (E) NE accumulation of ectopically expressed GFP-p150^Glued was quantified as in C. (Scale bars, 5 μm.)

Fig. 5.

Plk1 phosphorylation of p150^Glued promotes its dissociation from microtubules during early mitosis. (A and B) U2OS cells stably expressing GFP-p150^Glued constructs (WT, S179A, or S179D) were stained with α-tubulin antibodies. (C) Extracts from U2OS cells stably expressing GFP-p150^Glued constructs (WT, S179A, or S179D) were prepared for anti-GFP IP, followed by Western blotting with indicated antibodies. (D) After GST-p150^Glued-aa-1–310 protein bound on glutathione-agarose beads was incubated with or without purified Plk1 in the presence of ATP and microtubules were added for an additional 0.5 h. Microtubules associated with the GST-fusion proteins were isolated by low-speed centrifugation, separated by SDS/PAGE, and stained by Coomassie Brilliant Blue. (E) GST-p150^Glued-aa-1–310 (WT, S179A, or S179D) proteins bound on glutathione-agarose beads were incubated with microtubules and analyzed as in D. (Scale bars, 10 μm.)

Fig. 6.

Expression of the unphosphorylatable p150^Glued at Ser-179 retards NEBD in prophase. (A–D) U2OS cells stably expressing GFP-p150^Glued (WT, S179A, or S179D) were depleted of endogenous p150^Glued by RNAi, synchronized with the DTB, released for 11 h, and immunostained with lamin A/C antibodies. (B) Quantification of cells with NEBD. (C) Quantification of prophase cells with NE invaginations as in Fig. 3C. (D) Representative images of cells with two types of lamin A/C staining during NEBD. Although lamin A/C staining is completely undectable in “a,” a remaining lamin A/C signal is still clearly visible surrounding chromosomes in “b.” (Right) The ratio of b/(a+b) is shown. (Scale bars, 5 μm.)