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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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14-3-3 protein controls proper SAC inactivation in a γ-subtype-dependent manner.(a) Forty-eight hours after transfection with an indicated siRNA, HeLa cells were stained with anti-H3-pSer28 (red) and 4',6-diamidino-2-phenylindole (blue). Immunoblotting with anti-H3-pSer28 and anti-α-tubulin (loading control) are also shown in insets. (b) The effect of 14-3-3γ knockdown on cell-cycle progression after DTB synchronization. (c) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells. Box-and-whisker plots show the elapsed time that cells spent in mitosis from nuclear envelope breakdown (t=0) to anaphase onset (n≥60 from two independent experiments). (d) The effect of 14-3-3γ silencing on KT-associated BubR1. Scale bar, 10 μm. (e, f) The effects of BubR1 or Mad2 silencing on mitotic arrest induced by 14-3-3γ depletion. Mitotic indices are shown as mean±s.e.m. from three independent experiments (n>200 each). *P<0.01; Student’s t-test.
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f1: 14-3-3 protein controls proper SAC inactivation in a γ-subtype-dependent manner.(a) Forty-eight hours after transfection with an indicated siRNA, HeLa cells were stained with anti-H3-pSer28 (red) and 4',6-diamidino-2-phenylindole (blue). Immunoblotting with anti-H3-pSer28 and anti-α-tubulin (loading control) are also shown in insets. (b) The effect of 14-3-3γ knockdown on cell-cycle progression after DTB synchronization. (c) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells. Box-and-whisker plots show the elapsed time that cells spent in mitosis from nuclear envelope breakdown (t=0) to anaphase onset (n≥60 from two independent experiments). (d) The effect of 14-3-3γ silencing on KT-associated BubR1. Scale bar, 10 μm. (e, f) The effects of BubR1 or Mad2 silencing on mitotic arrest induced by 14-3-3γ depletion. Mitotic indices are shown as mean±s.e.m. from three independent experiments (n>200 each). *P<0.01; Student’s t-test.

Mentions: We previously reported that 14-3-3γ participates in the DNA damage response through the modulation of a signalling pathway that links Chk1 to Cdc25A1314. In order to examine whether 14-3-3 proteins are also involved in cell-cycle progression in the absence of exogenously introduced DNA damage, we examined the effects of 14-3-3 knockdown by transfection with short interfering RNAs (siRNAs) specific for each 14-3-3 subtype. For each protein, we targeted two independent sequences. Immunoblotting with antibodies against each subtype (characterized in Supplementary Fig. S1a) indicated the successful depletion of each subtype of 14-3-3 (Supplementary Fig. S1b). As judged by morphological features and mitotic marker phosphorylation (histone H3-Ser28 phosphorylation)15, 14-3-3γ depletion increased the mitotic index, whereas the depletion of other subtypes had only marginal effects (Fig. 1a). To examine this phenomenon more precisely, we combined siRNA transfection with double-thymidine block (DTB) synchronization16. In cells treated with control siRNA (siControl), the mitotic index reached a peak at 11 h after release from a second thymidine block and rapidly decreased thereafter. However, the decline in mitotic index was severely impaired in cells treated with 14-3-3γ-specific siRNA (si14-3-3γ), whereas we observed only marginal changes in the timing of mitotic entry and the height of the mitotic index peak (Fig. 1b). Next, we performed live-cell imaging assays using HeLa cells stably expressing histone H2B-GFP (green fluorescent protein)17. Control cells proceeded rapidly from nuclear envelope breakdown to anaphase onset (mean of 36 min; Fig. 1c and Supplementary Movie 1), whereas 14-3-3γ depletion yielded a significant increase in this duration (target sequence #1, mean of 153 min; #2, mean of 113 min; Fig. 1c and Supplementary Movie 2). These results suggested that 14-3-3γ depletion leads to a prometaphase/metaphase-like arrest.


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)

14-3-3 protein controls proper SAC inactivation in a γ-subtype-dependent manner.(a) Forty-eight hours after transfection with an indicated siRNA, HeLa cells were stained with anti-H3-pSer28 (red) and 4',6-diamidino-2-phenylindole (blue). Immunoblotting with anti-H3-pSer28 and anti-α-tubulin (loading control) are also shown in insets. (b) The effect of 14-3-3γ knockdown on cell-cycle progression after DTB synchronization. (c) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells. Box-and-whisker plots show the elapsed time that cells spent in mitosis from nuclear envelope breakdown (t=0) to anaphase onset (n≥60 from two independent experiments). (d) The effect of 14-3-3γ silencing on KT-associated BubR1. Scale bar, 10 μm. (e, f) The effects of BubR1 or Mad2 silencing on mitotic arrest induced by 14-3-3γ depletion. Mitotic indices are shown as mean±s.e.m. from three independent experiments (n>200 each). *P<0.01; Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: 14-3-3 protein controls proper SAC inactivation in a γ-subtype-dependent manner.(a) Forty-eight hours after transfection with an indicated siRNA, HeLa cells were stained with anti-H3-pSer28 (red) and 4',6-diamidino-2-phenylindole (blue). Immunoblotting with anti-H3-pSer28 and anti-α-tubulin (loading control) are also shown in insets. (b) The effect of 14-3-3γ knockdown on cell-cycle progression after DTB synchronization. (c) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells. Box-and-whisker plots show the elapsed time that cells spent in mitosis from nuclear envelope breakdown (t=0) to anaphase onset (n≥60 from two independent experiments). (d) The effect of 14-3-3γ silencing on KT-associated BubR1. Scale bar, 10 μm. (e, f) The effects of BubR1 or Mad2 silencing on mitotic arrest induced by 14-3-3γ depletion. Mitotic indices are shown as mean±s.e.m. from three independent experiments (n>200 each). *P<0.01; Student’s t-test.
Mentions: We previously reported that 14-3-3γ participates in the DNA damage response through the modulation of a signalling pathway that links Chk1 to Cdc25A1314. In order to examine whether 14-3-3 proteins are also involved in cell-cycle progression in the absence of exogenously introduced DNA damage, we examined the effects of 14-3-3 knockdown by transfection with short interfering RNAs (siRNAs) specific for each 14-3-3 subtype. For each protein, we targeted two independent sequences. Immunoblotting with antibodies against each subtype (characterized in Supplementary Fig. S1a) indicated the successful depletion of each subtype of 14-3-3 (Supplementary Fig. S1b). As judged by morphological features and mitotic marker phosphorylation (histone H3-Ser28 phosphorylation)15, 14-3-3γ depletion increased the mitotic index, whereas the depletion of other subtypes had only marginal effects (Fig. 1a). To examine this phenomenon more precisely, we combined siRNA transfection with double-thymidine block (DTB) synchronization16. In cells treated with control siRNA (siControl), the mitotic index reached a peak at 11 h after release from a second thymidine block and rapidly decreased thereafter. However, the decline in mitotic index was severely impaired in cells treated with 14-3-3γ-specific siRNA (si14-3-3γ), whereas we observed only marginal changes in the timing of mitotic entry and the height of the mitotic index peak (Fig. 1b). Next, we performed live-cell imaging assays using HeLa cells stably expressing histone H2B-GFP (green fluorescent protein)17. Control cells proceeded rapidly from nuclear envelope breakdown to anaphase onset (mean of 36 min; Fig. 1c and Supplementary Movie 1), whereas 14-3-3γ depletion yielded a significant increase in this duration (target sequence #1, mean of 153 min; #2, mean of 113 min; Fig. 1c and Supplementary Movie 2). These results suggested that 14-3-3γ depletion leads to a prometaphase/metaphase-like arrest.

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