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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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14-3-3γ binds to Plk1 and stimulates its catalytic activity.(a,b) Identification of proteins bound to GST-14-3-3γ specifically in mitotic extract. Mitosis- and 14-3-3γ-specific binding proteins (bands 1-4) are shown to the right (a). The presence of Plk1 was also checked by immunoblotting with anti-Plk1 (b). An asterisk or arrow indicates the position of GST-14-3-3γ or Plk1, respectively (b). (c) IP with anti-Plk1 from interphase (I) or mitotic (M) HeLa cell extract. The same amount of mouse IgG was used as a negative control. (d) GST pull-down assays using each subtype of GST-14-3-3 proteins form mitotic cell extract. (e–i) The effect of 14-3-3γ silencing on catalytic activities of mitotic kinases. Anti-Plk1 (e, i), anti-AurA (f), anti-AurB (g) or anti-Cyclin B1 immunoprecipitates (h) were subjected to IP kinase assays. Each immunoprecipitates and cell lysates (input) were also subjected to immunoblotting. In f, 8–128 ng μl−1 of purified 14-3-3γ was added in mitotic extract before IP. (j) The effects of 14-3-3γ or Plk1 depletion on intracellular phosphorylations.
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f2: 14-3-3γ binds to Plk1 and stimulates its catalytic activity.(a,b) Identification of proteins bound to GST-14-3-3γ specifically in mitotic extract. Mitosis- and 14-3-3γ-specific binding proteins (bands 1-4) are shown to the right (a). The presence of Plk1 was also checked by immunoblotting with anti-Plk1 (b). An asterisk or arrow indicates the position of GST-14-3-3γ or Plk1, respectively (b). (c) IP with anti-Plk1 from interphase (I) or mitotic (M) HeLa cell extract. The same amount of mouse IgG was used as a negative control. (d) GST pull-down assays using each subtype of GST-14-3-3 proteins form mitotic cell extract. (e–i) The effect of 14-3-3γ silencing on catalytic activities of mitotic kinases. Anti-Plk1 (e, i), anti-AurA (f), anti-AurB (g) or anti-Cyclin B1 immunoprecipitates (h) were subjected to IP kinase assays. Each immunoprecipitates and cell lysates (input) were also subjected to immunoblotting. In f, 8–128 ng μl−1 of purified 14-3-3γ was added in mitotic extract before IP. (j) The effects of 14-3-3γ or Plk1 depletion on intracellular phosphorylations.

Mentions: As 14-3-3 is known as a scaffold protein2021, we searched for binding partners of 14-3-3γ in mitosis. By the combination of glutathione S-transferase (GST) pull-down and mass spectrometry/mass spectrometry analyses, we identified several putative proteins, which bound to GST-14-3-3γ in a mitosis-specific manner (Fig. 2a). Among these proteins, we focused on Plk1 because the phenotype of 14-3-3γ depletion (Fig. 1) closely resembled that of Plk1 depletion and other identified proteins were already reported to interact with other subtypes of 14-3-32223. Immunoblotting with anti-Plk1 confirmed that Plk1 was indeed the 68-kDa protein brought down by GST-14-3-3γ from mitotic extract (Fig. 2b). GFP-14-3-3γ and Myc–Plk1 were also coimmunoprecipitated from cells transfected with these proteins (Supplementary Fig. S2a). At endogenous levels, the two proteins were coimmunoprecipitated from mitotic but not interphase cell extracts (Fig. 2c). Among seven 14-3-3 members analysed, Plk1 had the highest affinity for the γ-subtype (Fig. 2d), indicating that Plk1 was likely to be a genuine binding partner of 14-3-3γ during mitosis.


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)

14-3-3γ binds to Plk1 and stimulates its catalytic activity.(a,b) Identification of proteins bound to GST-14-3-3γ specifically in mitotic extract. Mitosis- and 14-3-3γ-specific binding proteins (bands 1-4) are shown to the right (a). The presence of Plk1 was also checked by immunoblotting with anti-Plk1 (b). An asterisk or arrow indicates the position of GST-14-3-3γ or Plk1, respectively (b). (c) IP with anti-Plk1 from interphase (I) or mitotic (M) HeLa cell extract. The same amount of mouse IgG was used as a negative control. (d) GST pull-down assays using each subtype of GST-14-3-3 proteins form mitotic cell extract. (e–i) The effect of 14-3-3γ silencing on catalytic activities of mitotic kinases. Anti-Plk1 (e, i), anti-AurA (f), anti-AurB (g) or anti-Cyclin B1 immunoprecipitates (h) were subjected to IP kinase assays. Each immunoprecipitates and cell lysates (input) were also subjected to immunoblotting. In f, 8–128 ng μl−1 of purified 14-3-3γ was added in mitotic extract before IP. (j) The effects of 14-3-3γ or Plk1 depletion on intracellular phosphorylations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: 14-3-3γ binds to Plk1 and stimulates its catalytic activity.(a,b) Identification of proteins bound to GST-14-3-3γ specifically in mitotic extract. Mitosis- and 14-3-3γ-specific binding proteins (bands 1-4) are shown to the right (a). The presence of Plk1 was also checked by immunoblotting with anti-Plk1 (b). An asterisk or arrow indicates the position of GST-14-3-3γ or Plk1, respectively (b). (c) IP with anti-Plk1 from interphase (I) or mitotic (M) HeLa cell extract. The same amount of mouse IgG was used as a negative control. (d) GST pull-down assays using each subtype of GST-14-3-3 proteins form mitotic cell extract. (e–i) The effect of 14-3-3γ silencing on catalytic activities of mitotic kinases. Anti-Plk1 (e, i), anti-AurA (f), anti-AurB (g) or anti-Cyclin B1 immunoprecipitates (h) were subjected to IP kinase assays. Each immunoprecipitates and cell lysates (input) were also subjected to immunoblotting. In f, 8–128 ng μl−1 of purified 14-3-3γ was added in mitotic extract before IP. (j) The effects of 14-3-3γ or Plk1 depletion on intracellular phosphorylations.
Mentions: As 14-3-3 is known as a scaffold protein2021, we searched for binding partners of 14-3-3γ in mitosis. By the combination of glutathione S-transferase (GST) pull-down and mass spectrometry/mass spectrometry analyses, we identified several putative proteins, which bound to GST-14-3-3γ in a mitosis-specific manner (Fig. 2a). Among these proteins, we focused on Plk1 because the phenotype of 14-3-3γ depletion (Fig. 1) closely resembled that of Plk1 depletion and other identified proteins were already reported to interact with other subtypes of 14-3-32223. Immunoblotting with anti-Plk1 confirmed that Plk1 was indeed the 68-kDa protein brought down by GST-14-3-3γ from mitotic extract (Fig. 2b). GFP-14-3-3γ and Myc–Plk1 were also coimmunoprecipitated from cells transfected with these proteins (Supplementary Fig. S2a). At endogenous levels, the two proteins were coimmunoprecipitated from mitotic but not interphase cell extracts (Fig. 2c). Among seven 14-3-3 members analysed, Plk1 had the highest affinity for the γ-subtype (Fig. 2d), indicating that Plk1 was likely to be a genuine binding partner of 14-3-3γ during mitosis.

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