Limits...
Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.

Smith E, Hégarat N, Vesely C, Roseboom I, Larch C, Streicher H, Straatman K, Flynn H, Skehel M, Hirota T, Kuriyama R, Hochegger H - EMBO J. (2011)

Bottom Line: Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927.Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation.Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, UK.

ABSTRACT
Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.

Show MeSH

Related in: MedlinePlus

Eg5 phosphorylation in G2 and M phase. (A) Immuno-blots probing extracts of cdk1as cells at different cell-cycle stages with the indicated antibodies. As: asynchronous cells; 1NM: cdk1as cells treated for 6 h with 1NMPP1; Mit: cells released for 30 min from the 1NMPP1 arrest; G1: cells released for 90 min from the 1NMPP1 arrest. (B) Immuno-blots with the indicated antibodies of DT40 extracts. cdk1as cells were treated with 10 μM 1NMPP1 (1NM) or co-treated with 10 μM 1NMPP1 and 100 nM BI2536 for 6 h (1NM/BI). (C) Kinase assays using bacterially expressed recombinant GST-Eg5 and Cdk2/cyclinA probed with Eg5, and phospho-specific Eg5 P-Thr 927 antibodies. (D) Immuno-blots probing extracts of DT40 cells with the indicated antibodies. cdk1as cells were treated for 6 h with 1NMPP1, and for an additional 2 h with or without 50 μM Roscovitine (1NM+ROS). (E) Immuno-blots probing extracts from cdk1as/cdk2 DT40 cells subjected to the indicated treatments. Cells in the final lane had been treated for 8 h with 0.5 μM 1NMPP1 and shifted to 10 μM 1NMPP1 for another 2 h. (F) Immuno-fluorescent staining of HeLa cells with Eg5 and Pericentrin antibodies. Cells were treated for 22 h with 10 μM RO3306 (RO), and 50 μM Roscovitine or 100 nM BI2536 were added for the last 2 h (RO+ROS and RO+BI). Graphs show a quantification of the signal intensity of either antibody at the centrosomes using ImageJ analysis tools (for each sample N>100).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3117641&req=5

f5: Eg5 phosphorylation in G2 and M phase. (A) Immuno-blots probing extracts of cdk1as cells at different cell-cycle stages with the indicated antibodies. As: asynchronous cells; 1NM: cdk1as cells treated for 6 h with 1NMPP1; Mit: cells released for 30 min from the 1NMPP1 arrest; G1: cells released for 90 min from the 1NMPP1 arrest. (B) Immuno-blots with the indicated antibodies of DT40 extracts. cdk1as cells were treated with 10 μM 1NMPP1 (1NM) or co-treated with 10 μM 1NMPP1 and 100 nM BI2536 for 6 h (1NM/BI). (C) Kinase assays using bacterially expressed recombinant GST-Eg5 and Cdk2/cyclinA probed with Eg5, and phospho-specific Eg5 P-Thr 927 antibodies. (D) Immuno-blots probing extracts of DT40 cells with the indicated antibodies. cdk1as cells were treated for 6 h with 1NMPP1, and for an additional 2 h with or without 50 μM Roscovitine (1NM+ROS). (E) Immuno-blots probing extracts from cdk1as/cdk2 DT40 cells subjected to the indicated treatments. Cells in the final lane had been treated for 8 h with 0.5 μM 1NMPP1 and shifted to 10 μM 1NMPP1 for another 2 h. (F) Immuno-fluorescent staining of HeLa cells with Eg5 and Pericentrin antibodies. Cells were treated for 22 h with 10 μM RO3306 (RO), and 50 μM Roscovitine or 100 nM BI2536 were added for the last 2 h (RO+ROS and RO+BI). Graphs show a quantification of the signal intensity of either antibody at the centrosomes using ImageJ analysis tools (for each sample N>100).

Mentions: We next investigated how Cdks and Plk1 regulate Eg5 in G2 and M phase. A key event in the initiation of centrosome separation is the phosphorylation of Eg5 in its C-terminal tail domain at Thr927 stimulating its binding to MTs and localization at the mitotic spindle (Blangy et al, 1995; Sawin and Mitchison, 1995; Cahu et al, 2008). Mitotic Cdk1 is thought to be responsible for this phosphorylation. However, we observed that Eg5 mediates centrosome separation independently of Cdk1. This prompted us to probe the phosphorylation of Eg5 after Cdk1 inhibition using a P-Thr927 phospho-specific antibody (Materials and methods). We confirmed that this antibody only cross-reacted with phosphorylated Eg5 (Supplementary Figure S3A and B) and used it to compare Eg5 phosphorylation in Cdk1-inhibited and mitotic cells (DT40 cdk1as cells in Figure 5A; HeLa cells in Supplementary Figure S3C). Surprisingly, we found that the majority of Eg5 was already phosphorylated at Thr927 despite Cdk1 inhibition in both human and chicken cells. There was no major increase in phosphorylation in the released mitotic cells, but phosphorylation levels dropped significantly as cells progressed into G1 phase (Figure 5A). Plk1 inhibition had no effect on Eg5 phosphorylation at Thr927 (Figure 5B) suggesting that the kinase is not involved in this control pathway.


Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.

Smith E, Hégarat N, Vesely C, Roseboom I, Larch C, Streicher H, Straatman K, Flynn H, Skehel M, Hirota T, Kuriyama R, Hochegger H - EMBO J. (2011)

Eg5 phosphorylation in G2 and M phase. (A) Immuno-blots probing extracts of cdk1as cells at different cell-cycle stages with the indicated antibodies. As: asynchronous cells; 1NM: cdk1as cells treated for 6 h with 1NMPP1; Mit: cells released for 30 min from the 1NMPP1 arrest; G1: cells released for 90 min from the 1NMPP1 arrest. (B) Immuno-blots with the indicated antibodies of DT40 extracts. cdk1as cells were treated with 10 μM 1NMPP1 (1NM) or co-treated with 10 μM 1NMPP1 and 100 nM BI2536 for 6 h (1NM/BI). (C) Kinase assays using bacterially expressed recombinant GST-Eg5 and Cdk2/cyclinA probed with Eg5, and phospho-specific Eg5 P-Thr 927 antibodies. (D) Immuno-blots probing extracts of DT40 cells with the indicated antibodies. cdk1as cells were treated for 6 h with 1NMPP1, and for an additional 2 h with or without 50 μM Roscovitine (1NM+ROS). (E) Immuno-blots probing extracts from cdk1as/cdk2 DT40 cells subjected to the indicated treatments. Cells in the final lane had been treated for 8 h with 0.5 μM 1NMPP1 and shifted to 10 μM 1NMPP1 for another 2 h. (F) Immuno-fluorescent staining of HeLa cells with Eg5 and Pericentrin antibodies. Cells were treated for 22 h with 10 μM RO3306 (RO), and 50 μM Roscovitine or 100 nM BI2536 were added for the last 2 h (RO+ROS and RO+BI). Graphs show a quantification of the signal intensity of either antibody at the centrosomes using ImageJ analysis tools (for each sample N>100).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117641&req=5

f5: Eg5 phosphorylation in G2 and M phase. (A) Immuno-blots probing extracts of cdk1as cells at different cell-cycle stages with the indicated antibodies. As: asynchronous cells; 1NM: cdk1as cells treated for 6 h with 1NMPP1; Mit: cells released for 30 min from the 1NMPP1 arrest; G1: cells released for 90 min from the 1NMPP1 arrest. (B) Immuno-blots with the indicated antibodies of DT40 extracts. cdk1as cells were treated with 10 μM 1NMPP1 (1NM) or co-treated with 10 μM 1NMPP1 and 100 nM BI2536 for 6 h (1NM/BI). (C) Kinase assays using bacterially expressed recombinant GST-Eg5 and Cdk2/cyclinA probed with Eg5, and phospho-specific Eg5 P-Thr 927 antibodies. (D) Immuno-blots probing extracts of DT40 cells with the indicated antibodies. cdk1as cells were treated for 6 h with 1NMPP1, and for an additional 2 h with or without 50 μM Roscovitine (1NM+ROS). (E) Immuno-blots probing extracts from cdk1as/cdk2 DT40 cells subjected to the indicated treatments. Cells in the final lane had been treated for 8 h with 0.5 μM 1NMPP1 and shifted to 10 μM 1NMPP1 for another 2 h. (F) Immuno-fluorescent staining of HeLa cells with Eg5 and Pericentrin antibodies. Cells were treated for 22 h with 10 μM RO3306 (RO), and 50 μM Roscovitine or 100 nM BI2536 were added for the last 2 h (RO+ROS and RO+BI). Graphs show a quantification of the signal intensity of either antibody at the centrosomes using ImageJ analysis tools (for each sample N>100).
Mentions: We next investigated how Cdks and Plk1 regulate Eg5 in G2 and M phase. A key event in the initiation of centrosome separation is the phosphorylation of Eg5 in its C-terminal tail domain at Thr927 stimulating its binding to MTs and localization at the mitotic spindle (Blangy et al, 1995; Sawin and Mitchison, 1995; Cahu et al, 2008). Mitotic Cdk1 is thought to be responsible for this phosphorylation. However, we observed that Eg5 mediates centrosome separation independently of Cdk1. This prompted us to probe the phosphorylation of Eg5 after Cdk1 inhibition using a P-Thr927 phospho-specific antibody (Materials and methods). We confirmed that this antibody only cross-reacted with phosphorylated Eg5 (Supplementary Figure S3A and B) and used it to compare Eg5 phosphorylation in Cdk1-inhibited and mitotic cells (DT40 cdk1as cells in Figure 5A; HeLa cells in Supplementary Figure S3C). Surprisingly, we found that the majority of Eg5 was already phosphorylated at Thr927 despite Cdk1 inhibition in both human and chicken cells. There was no major increase in phosphorylation in the released mitotic cells, but phosphorylation levels dropped significantly as cells progressed into G1 phase (Figure 5A). Plk1 inhibition had no effect on Eg5 phosphorylation at Thr927 (Figure 5B) suggesting that the kinase is not involved in this control pathway.

Bottom Line: Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927.Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation.Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, UK.

ABSTRACT
Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.

Show MeSH
Related in: MedlinePlus