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Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos.

Tavernier N, Noatynska A, Panbianco C, Martino L, Van Hove L, Schwager F, Léger T, Gotta M, Pintard L - J. Cell Biol. (2015)

Bottom Line: We further show that phospho-SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A.Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans.Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jacques Monod Institute, UMR7592; and Mass Spectrometry Facility, Jacques Monod Institute, UMR7592; Paris-Diderot University-Centre National de la Recherche Scientifique, 75013 Paris, France.

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SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[32P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.
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fig1: SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[32P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.

Mentions: SPAT-1 is a phosphoprotein modified at multiple residues observed as slower migrating bands on 1D gel separation (Fig. 1 A, lanes 1 and 2; Noatynska et al., 2010). Given that SPAT-1 is a PLK-1 substrate (Noatynska et al., 2010), these forms could correspond to species phosphorylated by PLK-1. However, SPAT-1–phosphorylated forms accumulated in plk-1 temperature-sensitive mutant embryos and in PLK-1–depleted embryos (Fig. 1 A, lanes 5 and 6; Noatynska et al., 2010), indicating that at least another kinase phosphorylates SPAT-1 in vivo.


Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos.

Tavernier N, Noatynska A, Panbianco C, Martino L, Van Hove L, Schwager F, Léger T, Gotta M, Pintard L - J. Cell Biol. (2015)

SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[32P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362466&req=5

fig1: SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[32P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.
Mentions: SPAT-1 is a phosphoprotein modified at multiple residues observed as slower migrating bands on 1D gel separation (Fig. 1 A, lanes 1 and 2; Noatynska et al., 2010). Given that SPAT-1 is a PLK-1 substrate (Noatynska et al., 2010), these forms could correspond to species phosphorylated by PLK-1. However, SPAT-1–phosphorylated forms accumulated in plk-1 temperature-sensitive mutant embryos and in PLK-1–depleted embryos (Fig. 1 A, lanes 5 and 6; Noatynska et al., 2010), indicating that at least another kinase phosphorylates SPAT-1 in vivo.

Bottom Line: We further show that phospho-SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A.Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans.Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jacques Monod Institute, UMR7592; and Mass Spectrometry Facility, Jacques Monod Institute, UMR7592; Paris-Diderot University-Centre National de la Recherche Scientifique, 75013 Paris, France.

Show MeSH
Related in: MedlinePlus