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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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Ser99 is a novel phosphorylation site required for Plk1 binding to 14-3-3γ.(a,b) The indicated Myc–Plk1 fragments were transiently expressed in HeLa cells. In b, Myc–Plk1 fragments (residues 1–371) were mutated at the indicated Ser/Thr to Ala. Each mitotic extract was subjected to GST pull-down assays using GST-14-3-3γ (c) Endogenous Plk1 was immunoprecipitated from interphase (I) or mitotic (M) extract. Before the immunoblotting, the immunoprecipitates were treated with or without λPPase. (d) Specificity of anti-Plk1-pS99 was analysed by the peptide competition assays. The position of heavy (h.c.) or light (l.c.) chain of anti-Plk1 is also indicated. (e) The effect of 14-3-3γ or Akt1/2 silencing on cell-cycle progression after DTB synchronization. (f) The effect of Bora silencing on Plk1 phosphorylation. (g–j) Myc–Plk1 WT or its variant was induced by the addition of doxycycline (Dox) in Tet-On HeLa cells (g, h). In j, Myc-tagged human Plk1 (WT), fly Polo or nematode PLK1 was transiently introduced in HeLa cells. Each anti-Myc immunoprecipitate from mitotic cells was subjected to immunoblotting or IP kinase assays. Fold activation relative to Plk1 WT is shown in the graph. The alignment of Plk1 protein sequences around Ser99 is shown in i.
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f3: Ser99 is a novel phosphorylation site required for Plk1 binding to 14-3-3γ.(a,b) The indicated Myc–Plk1 fragments were transiently expressed in HeLa cells. In b, Myc–Plk1 fragments (residues 1–371) were mutated at the indicated Ser/Thr to Ala. Each mitotic extract was subjected to GST pull-down assays using GST-14-3-3γ (c) Endogenous Plk1 was immunoprecipitated from interphase (I) or mitotic (M) extract. Before the immunoblotting, the immunoprecipitates were treated with or without λPPase. (d) Specificity of anti-Plk1-pS99 was analysed by the peptide competition assays. The position of heavy (h.c.) or light (l.c.) chain of anti-Plk1 is also indicated. (e) The effect of 14-3-3γ or Akt1/2 silencing on cell-cycle progression after DTB synchronization. (f) The effect of Bora silencing on Plk1 phosphorylation. (g–j) Myc–Plk1 WT or its variant was induced by the addition of doxycycline (Dox) in Tet-On HeLa cells (g, h). In j, Myc-tagged human Plk1 (WT), fly Polo or nematode PLK1 was transiently introduced in HeLa cells. Each anti-Myc immunoprecipitate from mitotic cells was subjected to immunoblotting or IP kinase assays. Fold activation relative to Plk1 WT is shown in the graph. The alignment of Plk1 protein sequences around Ser99 is shown in i.

Mentions: To examine the binding mode of the two proteins, we purified GST–Plk1 from insect cells and 14-3-3γ from Escherichia coli: GST–Plk1 was already phosphorylated at several sites including Thr210 in insect cells (Supplementary Fig. S2b). GST pull-down assays revealed a direct interaction between the two purified proteins, Plk1 and 14-3-3γ (Supplementary Fig. S2b). Remarkably, treatment of GST–Plk1 with λ protein phosphatase (λPPase) completely abolished the binding between the two proteins (Supplementary Fig. S2b). Thus, we mutated reported Plk1 phosphorylation sites, Ser137, Thr210 (ref. 26) and/or Ser326 (ref. 27) to Ala and then transfected cells with these Plk1 mutants. However, even mutation of all three sites (3A) did not inhibit the binding to GST-14-3-3γ (Supplementary Fig. S2c). As the N-terminal portion (residues 1–201) of Plk1 was sufficient for the binding to 14-3-3γ (Fig. 3a), we constructed a series of Plk1 fragments (1–371) mutated at Ser or Thr residues within 1–201. As shown in Fig. 3b, Ala mutation at Ser99 specifically abolished the binding to GST-14-3-3γ, suggesting that Ser99 was the most likely candidate phosphorylation site required for 14-3-3γ binding.


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)

Ser99 is a novel phosphorylation site required for Plk1 binding to 14-3-3γ.(a,b) The indicated Myc–Plk1 fragments were transiently expressed in HeLa cells. In b, Myc–Plk1 fragments (residues 1–371) were mutated at the indicated Ser/Thr to Ala. Each mitotic extract was subjected to GST pull-down assays using GST-14-3-3γ (c) Endogenous Plk1 was immunoprecipitated from interphase (I) or mitotic (M) extract. Before the immunoblotting, the immunoprecipitates were treated with or without λPPase. (d) Specificity of anti-Plk1-pS99 was analysed by the peptide competition assays. The position of heavy (h.c.) or light (l.c.) chain of anti-Plk1 is also indicated. (e) The effect of 14-3-3γ or Akt1/2 silencing on cell-cycle progression after DTB synchronization. (f) The effect of Bora silencing on Plk1 phosphorylation. (g–j) Myc–Plk1 WT or its variant was induced by the addition of doxycycline (Dox) in Tet-On HeLa cells (g, h). In j, Myc-tagged human Plk1 (WT), fly Polo or nematode PLK1 was transiently introduced in HeLa cells. Each anti-Myc immunoprecipitate from mitotic cells was subjected to immunoblotting or IP kinase assays. Fold activation relative to Plk1 WT is shown in the graph. The alignment of Plk1 protein sequences around Ser99 is shown in i.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Ser99 is a novel phosphorylation site required for Plk1 binding to 14-3-3γ.(a,b) The indicated Myc–Plk1 fragments were transiently expressed in HeLa cells. In b, Myc–Plk1 fragments (residues 1–371) were mutated at the indicated Ser/Thr to Ala. Each mitotic extract was subjected to GST pull-down assays using GST-14-3-3γ (c) Endogenous Plk1 was immunoprecipitated from interphase (I) or mitotic (M) extract. Before the immunoblotting, the immunoprecipitates were treated with or without λPPase. (d) Specificity of anti-Plk1-pS99 was analysed by the peptide competition assays. The position of heavy (h.c.) or light (l.c.) chain of anti-Plk1 is also indicated. (e) The effect of 14-3-3γ or Akt1/2 silencing on cell-cycle progression after DTB synchronization. (f) The effect of Bora silencing on Plk1 phosphorylation. (g–j) Myc–Plk1 WT or its variant was induced by the addition of doxycycline (Dox) in Tet-On HeLa cells (g, h). In j, Myc-tagged human Plk1 (WT), fly Polo or nematode PLK1 was transiently introduced in HeLa cells. Each anti-Myc immunoprecipitate from mitotic cells was subjected to immunoblotting or IP kinase assays. Fold activation relative to Plk1 WT is shown in the graph. The alignment of Plk1 protein sequences around Ser99 is shown in i.
Mentions: To examine the binding mode of the two proteins, we purified GST–Plk1 from insect cells and 14-3-3γ from Escherichia coli: GST–Plk1 was already phosphorylated at several sites including Thr210 in insect cells (Supplementary Fig. S2b). GST pull-down assays revealed a direct interaction between the two purified proteins, Plk1 and 14-3-3γ (Supplementary Fig. S2b). Remarkably, treatment of GST–Plk1 with λ protein phosphatase (λPPase) completely abolished the binding between the two proteins (Supplementary Fig. S2b). Thus, we mutated reported Plk1 phosphorylation sites, Ser137, Thr210 (ref. 26) and/or Ser326 (ref. 27) to Ala and then transfected cells with these Plk1 mutants. However, even mutation of all three sites (3A) did not inhibit the binding to GST-14-3-3γ (Supplementary Fig. S2c). As the N-terminal portion (residues 1–201) of Plk1 was sufficient for the binding to 14-3-3γ (Fig. 3a), we constructed a series of Plk1 fragments (1–371) mutated at Ser or Thr residues within 1–201. As shown in Fig. 3b, Ala mutation at Ser99 specifically abolished the binding to GST-14-3-3γ, suggesting that Ser99 was the most likely candidate phosphorylation site required for 14-3-3γ binding.

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