Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint

Abstract

The spindle checkpoint ensures the accuracy of chromosome segregation during mitosis. The protein serine/threonine kinase, Mps1, is a critical component of the spindle checkpoint in human cells and regulates the kinetochore localization of key checkpoint proteins. The kinase activity of Mps1 is required for the spindle checkpoint, but how Mps1 is activated during mitosis is unclear. Here, we show that the endogenous Mps1 in mitotic HeLa cells is phosphorylated on T676, a residue in the activation loop. This phosphorylation event on Mps1 is required for its kinase activity in vitro and for spindle checkpoint signaling in vivo. T676 phosphorylation of Mps1 increases during mitosis and can occur through intermolecular/trans autophosphorylation. Induced dimerization of Mps1 is sufficient to activate its kinase activity in cells. We speculate that the kinetochore localization of Mps1 raises its local concentration, leading to its activation during mitosis through more efficient trans autophosphorylation.

Fig. 1.

Identification of phosphorylation sites in human Mps1 protein. (A) In vivo phosphorylation sites of Mps1 from mitotic HeLa cells identified by mass spectrometry. (B) Tandem mass spectrum of the phospho-T676-containing peptide. “b” and “y” ion series represent fragment ions containing the N- and C-termini of the peptide, respectively. Ions labeled with “-P” indicate a neutral loss of H₃PO₄, whereas ions labeled with “*” indicate a neutral loss of NH₃. (C) Sequence alignment of the activation loops of Mps1 proteins from Homo sapiens (Hs), Mus musculus (Mm), Xenopus laevis (Xl), Drosophila melanogaster (Dm), Schizosaccharomyces pombe (Sp), and Saccharomyces cerevisiae (Sc). T676 and the DFG motif that binds Mg²⁺-ATP are shown in bold. (D) A structure model of the kinase domain of human Mps1. The activation loop is shown in yellow. ATP and several residues discussed in the text are shown as sticks.

Fig. 2.

Autophosphorylation of T676 is required for the kinase activity of Mps1. (A) In vitro translated Myc-Mps1 was IPed by using anti-Myc beads and incubated with 5 μg of MBP in the presence of γ-[³²P]ATP for 30 min at 30°C. The reaction mixture was separated on SDS/PAGE followed by blotting with anti-Mps1 (Top), autoradiography (Middle), and Coomassie blue staining (Bottom). (B) Approximately 60 ng of the wild-type (WT) or 300 ng of kinase-dead mutant (KD) of GST-Mps1 IPed from Sf9 cell lysate was either untreated or treated with λ phosphatase and assayed for its kinase activity against 5 μg of MBP with or without a preincubation with cold ATP. The reaction mixture was separated on SDS/PAGE and analyzed by autoradiography (Top and Middle) and Coomassie blue staining (Bottom). (C) Approximately 60 ng of the WT or 300 ng of KD of GST-Mps1 was IPed from Sf9 cell lysate by using anti-Mps1 beads and incubated with 2 μg of bacterially expressed C-terminal fragment of Mps1 (Mps1C) in the presence of γ-[³²P]ATP for 30 min at 30°C. The reaction mixture was separated on SDS/PAGE and analyzed by autoradiography (Upper) and Coomassie blue staining (Lower).

Fig. 3.

The Mps1 T676A mutant is defective in spindle-checkpoint function. (A) HeLa tet-on cells were transfected with Mps1 siRNA and the indicated RNAi-resistant Mps1 vectors and then treated with nocodazole. The cell lysates were blotted with the indicated antibodies. The positions of Myc-Mps1 and the endogenous (Endo.) Mps1 are indicated. (B) The mitotic indices of the cells described in A. Approximately 1,000 cells from three independent experiments were counted. The averages and standard deviations are shown. (C) HeLa tet-on cells were transfected with Mps1 siRNA and the indicated RNAi-resistant Mps1 vectors. After incubation with nocodazole for 16 h, the cells were trypsinized, spun down on slides, fixed, and stained with anti-phospho-Histone H3 (red), anti-Myc (green), and DAPI (blue). (D) The mitotic indices of Myc-positive cells described in C. More than 100 cells expressing Myc-Mps1 in two experiments were counted. The average and standard deviations are shown.

Fig. 4.

The kinetochore localization of BubR1 is defective in Mps1 RNAi cells expressing Mps1 T676A. HeLa tet-on cells were transfected with Mps1 siRNA and the indicated RNAi-resistant Mps1 vectors. The cells were stained with anti-Myc, CREST (red), anti-BubR1 (green), and DAPI (blue). (Scale bars, 5 μm.)

Fig. 5.

T676 phosphorylation of Mps1 increases during mitosis. (A) The endogenous Mps1 was IPed from thymidine (Thy)- or nocodazole (Noc)-arrested HeLa tet-on cells and incubated with MBP and γ-[³²P]ATP in the presence or absence of the Mps1 inhibitor, SP600125. The reaction mixture was separated on SDS/PAGE and analyzed by autoradiography (Top), Coomassie blue staining (Middle), or anti-Mps1 blotting (Bottom). (B) Phosphopeptide analysis of in vitro translated Myc-Mps1 KD and KD/676A that were phosphorylated by GST-Mps1 in the presence of γ-[³²P]ATP. The chromatography and electrophoresis dimensions are labeled. The open circles indicate the sample origin for electrophoresis. The position of the phosphopeptide containing T676 of Mps1 is indicated by an arrow. (C) Phosphopeptide analysis of the endogenous Mps1 that was IPed from G₁/S or mitotic HeLa cells metabolically labeled with γ-[³²P]-orthophosphate. Phosphopeptides absent in Mps1 from G₁/S cells are indicated by arrows.

Fig. 6.

Induced dimerization of Mps1 increases its kinase activity in cells. (A) Schematic drawing of the FKBP-Mps1 fusion protein and its chemical-induced dimerization. (B) HeLa tet-on cells were transfected with plasmids encoding Myc-FKBP-Mps1 WT or KD and incubated with or without AP20187. The total cell lysate was blotted with anti-Myc. (C) The Myc-FKBP-Mps1 proteins were IPed from lysates of cells described in B and assayed for their kinase activity against MBP in the presence of γ-[³²P]ATP. The reaction mixtures were separated on SDS/PAGE and analyzed by autoradiography, anti-Myc blotting, or Coomassie blue staining.