Phosphorylation- and polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1

Abstract

Polo-like kinase 1 (Plk1) is required for the generation of the tension-sensing 3F3/2 kinetochore epitope and facilitates kinetochore localization of Mad2 and other spindle checkpoint proteins. Here, we investigate the mechanism by which Plk1 itself is recruited to kinetochores. We show that Plk1 binds to budding uninhibited by benzimidazole 1 (Bub1) in mitotic human cells. The Plk1-Bub1 interaction requires the polo-box domain (PBD) of Plk1 and is enhanced by cyclin-dependent kinase 1 (Cdk1)-mediated phosphorylation of Bub1 at T609. The PBD-dependent binding of Plk1 to Bub1 facilitates phosphorylation of Bub1 by Plk1 in vitro. Depletion of Bub1 in HeLa cells by RNA interference (RNAi) diminishes the kinetochore localization of Plk1. Ectopic expression of the wild-type Bub1, but not the Bub1-T609A mutant, in Bub1-RNAi cells restores the kinetochore localization of Plk1. Our results suggest that phosphorylation of Bub1 at T609 by Cdk1 creates a docking site for the PBD of Plk1 and facilitates the kinetochore recruitment of Plk1.

Figure 1.

Bub1 and Plk1 interact and colocalize at the kinetochores in mitosis. (A) Lysate of nocodazole-arrested mitotic HeLa cells was immunoprecipitated with control IgG or α-Bub1. The IPs were separated on SDS-PAGE and stained with silver. (B) Lysates of HeLa cells treated with thymidine (Thy) or nocodazole (Noc) were immunoprecipitated with α-Bub1. The lysates and α-Bub1 IP were blotted with α-Bub1 and α-Plk1. (C) HeLa cells were transfected with plasmids encoding Myc-Bub1 together with HA-Plk1 or HA-Plk1-PBD (PBD). The cell lysates and the α-Myc IP were blotted with α-Myc and α-HA. (D) A HeLa cell at prometaphase was stained with α-Plk1 (green), α-Bub1 (red), and DAPI (blue). The boxed areas are magnified and shown in insets. Bar, 10 μm.

Figure 2.

The PBD of Plk1 mediates the binding between Plk1 and Bub1. (A) Schematic drawing of the domain structure of Plk1 and the locations of two critical phosphate-binding residues. (B) HeLa cells were transfected with plasmids encoding Myc-Bub1 together with HA-Plk1-WT, HA-Plk1-K82R (kinase-inactive mutant), or HA-Plk1-H538A/K540M and arrested in mitosis with nocodazole. The cell lysates and α-Myc IP were blotted with α-Myc and α-HA. (C) HeLa cells were transfected with plasmids encoding Myc-Bub1 and arrested in mitosis with nocodazole. The cell lysates were incubated with glutathione-agarose beads that contained GST, GST-PBD-WT, or GST-PBD-H538A/K540M. After washing, the proteins bound to beads were blotted with α-Myc (top) or stained with Coomassie blue (bottom).

Figure 3.

Phosphorylation of Bub1 at T609 occurs in vivo and is required for the Plk1–Bub1 interaction. (A) Sequence alignment of the two S-S/T-P motifs in human Bub1 (hBub1), mouse Bub1 (mBub1), Xenopus Bub1 (xBub1), human BubR1 (hBubR1), and mouse BubR1 (mBubR1). (B) HeLa cells were transfected with plasmids encoding Myc-Bub1-WT, Myc-Bub1-S99A, or Myc-Bub1-T609A and treated with thymidine (Thy) or nocodazole (Noc). The α-Myc IP of lysates from the transfected cells was blotted with the MPM-2 antibody (top) and α-Myc (bottom). (C) HeLa cells were transfected with plasmids encoding Myc-Bub1-WT, Myc-Bub1-T609A, Myc-Bub1-7A, or Myc-Bub1-6A-T609 and treated with Thy or Noc. The α-Myc IP of lysates from the transfected cells was blotted with the MPM-2 antibody (top) and α-Myc (bottom). (D) Lysates of HeLa cells treated with thymidine or nocodazole were IPed with α-GST or α-Bub1. The IPs were blotted with the MPM-2 antibody (left) and α-Bub1 (right). (E) HeLa cells were transfected with plasmids encoding HA-Plk1 together with Myc-Bub1-WT, Myc-Bub1-S99A, or Myc-Bub1-T609A and treated with nocodazole. The lysates and α-Myc IP from the transfected cells were blotted with α-HA and α-Myc.

Figure 4.

Cdk1 phosphorylates Bub1 and promotes Plk1-binding and Plk1-mediated phosphorylation of Bub1. (A) Recombinant purified Bub1-ΔKD (a Bub1 truncation mutant with its kinase domain deleted) was incubated with [γ-³²P]ATP and cyclin B1/Cdk1 in the absence or presence of 10 μM roscovitine for 30 min at room temperature. The reaction was quenched and analyzed by SDS-PAGE followed with autoradiography. Bottom, Coomassie-stained gel of the same reactions to indicate that equal amounts of Bub1-ΔKD were included in the reactions. (B) Two major in vitro Cdk1 phosphorylation sites on recombinant Bub1 mapped by mass spectrometry. (C) The Bub1-ΔKD protein was incubated with Cdk1 in the presence or absence of ATP. The reaction mixtures were incubated with glutathione-agarose beads that contained GST, GST-PBD-WT, or GST-PBD-H538A/K540M. After washing, the proteins bound to beads were blotted with α-Bub1. Bottom, Coomassie-stained gel of the same reactions to indicate that similar amounts of GST, GST-PBD-WT, and GST-PBD-H538A/K540M were present in the reactions. (C) Bub1-ΔKD and mutant proteins were incubated with Cdk1 in the kinase buffer containing nonradioactive ATP for 1 h at room temperature. The Bub1 proteins were then immunoprecipitated and subjected to a second kinase reaction with purified Plk1-T210D and [γ-³²P]ATP. After 1 h, the reactions were quenched and analyzed by SDS-PAGE followed by autoradiography (top). The same samples were blotted with α-Bub1 to show that similar amounts of Bub1 proteins were present (bottom). (D) Recombinant Bub1-ΔKD or Bub1-ΔKD-T609A from Sf9 cells were either untreated or treated with λ-phosphatase and blotted with MPM-2. (E) Nocodazole-arrested HeLa cells were incubated with 50 μM roscovitine for the indicated times and then lysed and subjected to α-Bub1 IP. The IPs were blotted with the MPM-2 antibody (top), α-Bub1 (middle), and α-Plk1 (bottom).

Figure 5.

Bub1-RNAi diminishes the kinetochore localization of Plk1. (A) HeLa cells that were either mock transfected or transfected with siRNAs against Bub1 or Plk1 were fixed and stained with α-Plk1, α-Bub1, CREST, and DAPI. In the merge, DAPI is pseudocolored blue, CREST in red, and Plk1 (top two panels) or Bub1 (bottom) in green. Bar, 5 μm. (B) HeLa cells that were either mock transfected or transfected with siRNA against Bub1 were fixed and stained with α-Plk1, CREST, γ-tubulin, and DAPI. In the merge, DAPI is pseudocolored blue, γ-tubulin red, and Plk1 green. The centrosomes are marked by arrows. Bar, 5 μm. (C) The kinetochore signal of Bub1 was quantified in mock, Plk1- and BubR1-RNAi cells. Kinetochores from more than 10 mitotic cells were analyzed and normalized using the CREST staining. The mean and SD are shown. (D) The kinetochore staining of Plk1 was quantified in mock, Bub1-, and BubR1-RNAi cells. Kinetochores from more than 10 mitotic cells were analyzed and normalized using the CREST staining. The mean and SD are shown.

Figure 6.

Expression of Bub1-T609A fails to restore the kinetochore localization of Plk1 in Bub1-RNAi cells. (A) HeLa Tet-On cells stably transfected with pTRE2-Myc-Bub1-WT or pTRE2-Myc-Bub1-T609A were cultured in the presence of doxycycline and treated with mock or Bub1-RNAi. The cell lysates were blotted with α-Bub1 and α-APC2. The positions of the endogenous (Endo) Bub1 and Myc-Bub1 are indicated. (B) The cells described in A were stained with α-Plk1 (green), α-Myc (red), and DAPI (blue). Bar, 5 μm. (C) The cells described in A were stained with α-Sgo1 (green), α-Myc (red), and DAPI (blue). (D) Quantification of Plk1 immunofluorescence signals at the kinetochores. (E) Quantification of Sgo1 immunofluorescence signals at the kinetochores.

Figure 7.

INCENP localizes to inner centromeres and is required for the kinetochore localization of Bub1 and Plk1. (A) HeLa cells that were either mock transfected or transfected with siRNA against INCENP were fixed and stained with α-Plk1, α-INCENP, CREST, and DAPI. In the merge, Plk1 staining is in green, INCENP staining is in red, and DAPI staining is in blue. The boxed areas are magnified and shown in insets. Bar, 5 μm. (B) HeLa cells that were either mock transfected or transfected with siRNA against INCENP were fixed and stained with α-Bub1, α-INCENP, CREST, and DAPI. In the merge, Bub1 staining is in green, INCENP staining is in red, and DAPI staining is in blue. The boxed areas are magnified and shown in insets. Bar, 5 μm. (C) Quantification of Plk1 immunofluorescence signals at the kinetochores in cells described in A. (D) Quantification of Bub1 immunofluorescence signals at the kinetochores in cells described in B. (E) Lysates of cells described in A were blotted with α-INCENP and α-APC2.