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PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3γ and is required for metaphase-anaphase transition.

Kasahara K, Goto H, Izawa I, Kiyono T, Watanabe N, Elowe S, Nigg EA, Inagaki M - Nat Commun (2013)

Bottom Line: Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition.Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner.Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.

ABSTRACT
Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1-Thr210 phosphorylation. Plk1-Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.

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Plk1–Ser99 phosphorylation is dispensable for mitotic entry after release from a second block.(a) The evaluation of Plk1 silencing using each target sequence of Plk1 (#1 or 3′-untranslated region (UTR)). (b) The effect of Plk1 silencing on mitotic entry after DTB synchronization. (c) The evaluation of mitotic index after the replacement. (d) The effect of Plk1 silencing on spindle bipolarity. Examples of monopolar or bipolar spindle formation are shown in the left. (e, f) The effect of the replacement with the indicated Plk1 (e) or 14-3-3γ silencing (f) on bipolar spindle formation. Data were obtained from two independent experiments (n>100; d–f). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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f5: Plk1–Ser99 phosphorylation is dispensable for mitotic entry after release from a second block.(a) The evaluation of Plk1 silencing using each target sequence of Plk1 (#1 or 3′-untranslated region (UTR)). (b) The effect of Plk1 silencing on mitotic entry after DTB synchronization. (c) The evaluation of mitotic index after the replacement. (d) The effect of Plk1 silencing on spindle bipolarity. Examples of monopolar or bipolar spindle formation are shown in the left. (e, f) The effect of the replacement with the indicated Plk1 (e) or 14-3-3γ silencing (f) on bipolar spindle formation. Data were obtained from two independent experiments (n>100; d–f). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Mentions: As Plk1 was also reported to have important roles in entry into mitosis after checkpoint recovery1011, we analysed the mitotic index after release from DTB-induced arrest. Strong silencing of Plk1 expression (siPlk1, 3′-untranslated region) induced a delay in mitotic entry, whereas weaker Plk1 depletion (siPlk1, #1) had only a marginal effect (Fig. 5a). This delay in mitotic entry was reverted by the expression of S99A, as well as WT Plk1 but not a Thr210-phospho-blocking mutant (T210A) or an ATP-binding-site mutant (K82R; Fig. 5c). Importantly, these latter mutants almost completely lacked catalytic activity, whereas Plk1–S99A activity was weak but detectable (Supplementary Fig. S3c; also see Fig. 3h). Similar effects on the kinetics of mitotic entry were observed when using nocodazole to arrest cells through SAC activation (Supplementary Fig. S3d). Next, we examined the functionality of Plk1 mutants during centrosome separation. Monopolar spindle formation was increased by strong Plk1 depletion (3′-untranslated region) but only marginally by partial depletion (#1; Fig. 5a). This monopolar spindle formation was reverted by the induction of S99A, as well as WT but not T210A or K82R versions of Plk1 (Fig. 5e). Consistent with these results, 14-3-3γ depletion had only marginal effects on the kinetics of mitotic entry after DTB-induced arrest (Fig. 1b) or the extent of bipolar spindle formation (Fig. 5f). These results suggested that Plk1 binding to 14-3-3γ through Plk1–Ser99 phosphorylation is dispensable for mitotic entry after checkpoint recovery, as well as for centrosome separation. They support and extend a previous study demonstrating that different mitotic functions of Plk1 show differential sensitivity to Plk1 regulatory mechanisms32.


PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3γ and is required for metaphase-anaphase transition.

Kasahara K, Goto H, Izawa I, Kiyono T, Watanabe N, Elowe S, Nigg EA, Inagaki M - Nat Commun (2013)

Plk1–Ser99 phosphorylation is dispensable for mitotic entry after release from a second block.(a) The evaluation of Plk1 silencing using each target sequence of Plk1 (#1 or 3′-untranslated region (UTR)). (b) The effect of Plk1 silencing on mitotic entry after DTB synchronization. (c) The evaluation of mitotic index after the replacement. (d) The effect of Plk1 silencing on spindle bipolarity. Examples of monopolar or bipolar spindle formation are shown in the left. (e, f) The effect of the replacement with the indicated Plk1 (e) or 14-3-3γ silencing (f) on bipolar spindle formation. Data were obtained from two independent experiments (n>100; d–f). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675326&req=5

f5: Plk1–Ser99 phosphorylation is dispensable for mitotic entry after release from a second block.(a) The evaluation of Plk1 silencing using each target sequence of Plk1 (#1 or 3′-untranslated region (UTR)). (b) The effect of Plk1 silencing on mitotic entry after DTB synchronization. (c) The evaluation of mitotic index after the replacement. (d) The effect of Plk1 silencing on spindle bipolarity. Examples of monopolar or bipolar spindle formation are shown in the left. (e, f) The effect of the replacement with the indicated Plk1 (e) or 14-3-3γ silencing (f) on bipolar spindle formation. Data were obtained from two independent experiments (n>100; d–f). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Mentions: As Plk1 was also reported to have important roles in entry into mitosis after checkpoint recovery1011, we analysed the mitotic index after release from DTB-induced arrest. Strong silencing of Plk1 expression (siPlk1, 3′-untranslated region) induced a delay in mitotic entry, whereas weaker Plk1 depletion (siPlk1, #1) had only a marginal effect (Fig. 5a). This delay in mitotic entry was reverted by the expression of S99A, as well as WT Plk1 but not a Thr210-phospho-blocking mutant (T210A) or an ATP-binding-site mutant (K82R; Fig. 5c). Importantly, these latter mutants almost completely lacked catalytic activity, whereas Plk1–S99A activity was weak but detectable (Supplementary Fig. S3c; also see Fig. 3h). Similar effects on the kinetics of mitotic entry were observed when using nocodazole to arrest cells through SAC activation (Supplementary Fig. S3d). Next, we examined the functionality of Plk1 mutants during centrosome separation. Monopolar spindle formation was increased by strong Plk1 depletion (3′-untranslated region) but only marginally by partial depletion (#1; Fig. 5a). This monopolar spindle formation was reverted by the induction of S99A, as well as WT but not T210A or K82R versions of Plk1 (Fig. 5e). Consistent with these results, 14-3-3γ depletion had only marginal effects on the kinetics of mitotic entry after DTB-induced arrest (Fig. 1b) or the extent of bipolar spindle formation (Fig. 5f). These results suggested that Plk1 binding to 14-3-3γ through Plk1–Ser99 phosphorylation is dispensable for mitotic entry after checkpoint recovery, as well as for centrosome separation. They support and extend a previous study demonstrating that different mitotic functions of Plk1 show differential sensitivity to Plk1 regulatory mechanisms32.

Bottom Line: Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition.Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner.Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.

ABSTRACT
Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1-Thr210 phosphorylation. Plk1-Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.

Show MeSH
Related in: MedlinePlus