Limits...
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.

Show MeSH

Related in: MedlinePlus

CyclinB/Cdk1 phosphorylates SPAT-1 at multiple sites. (A) Flow chart of the approach used to map SPAT-1 phosphorylation sites in vitro and in vivo. IP, immunoprecipitation. LC-MS/MS, tandem MS. (B) Table showing the amino acid position of the CDK-1–dependent phosphorylation sites in SPAT-1 protein sequence (F57C2.6b) with the 13 (S/T)p sites identified in vitro (highlighted in light gray), seven of which were confirmed in vivo (highlighted in dark gray). (C) Kinase assay conducted with purified Strep–SPAT-1 WT or Strep–SPAT-1 13A with human CyclinB/Cdk1 and γ-[32P]ATP. (top) SDS-PAGE gel stained with Coomassie shows protein loading. (bottom) Autoradiography indicates SPAT-1 phosphorylation status. (D) Strep pull-downs from insect Sf9 cells coexpressing PLK-1 and Strep–SPAT-1 WT (lanes 1 and 4), PLK-1 and SPAT-1 13A mutant (lanes 2 and 5), or PLK-1 alone (lanes 3 and 6) were analyzed by SDS-PAGE and probed with PLK-1 (top) and SPAT-1 (bottom) antibodies. A fraction (1/100) of the total protein extracts (Total) was loaded in the lanes 1–3. (E) Strep–SPAT-1 WT or the nonphosphorylatable 13A mutant version was immobilized on Strep-Tactin Sepharose beads, phosphorylated by CyclinB/Cdk1, and incubated with PLK-1 for pull-down experiments. The SPAT-1–PLK-1 complex was eluted with desthiobiotin (Elutions) and analyzed by SDS-PAGE. The membrane was blotted with PLK-1 (top) and SPAT-1 antibodies (bottom). (F) Western blot showing the result of the gel filtration analysis of Strep–SPAT-1 WT (top) and Strep–SPAT-1 13A (bottom) proteins. The proteins used to calibrate the column are indicated on the top.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: CyclinB/Cdk1 phosphorylates SPAT-1 at multiple sites. (A) Flow chart of the approach used to map SPAT-1 phosphorylation sites in vitro and in vivo. IP, immunoprecipitation. LC-MS/MS, tandem MS. (B) Table showing the amino acid position of the CDK-1–dependent phosphorylation sites in SPAT-1 protein sequence (F57C2.6b) with the 13 (S/T)p sites identified in vitro (highlighted in light gray), seven of which were confirmed in vivo (highlighted in dark gray). (C) Kinase assay conducted with purified Strep–SPAT-1 WT or Strep–SPAT-1 13A with human CyclinB/Cdk1 and γ-[32P]ATP. (top) SDS-PAGE gel stained with Coomassie shows protein loading. (bottom) Autoradiography indicates SPAT-1 phosphorylation status. (D) Strep pull-downs from insect Sf9 cells coexpressing PLK-1 and Strep–SPAT-1 WT (lanes 1 and 4), PLK-1 and SPAT-1 13A mutant (lanes 2 and 5), or PLK-1 alone (lanes 3 and 6) were analyzed by SDS-PAGE and probed with PLK-1 (top) and SPAT-1 (bottom) antibodies. A fraction (1/100) of the total protein extracts (Total) was loaded in the lanes 1–3. (E) Strep–SPAT-1 WT or the nonphosphorylatable 13A mutant version was immobilized on Strep-Tactin Sepharose beads, phosphorylated by CyclinB/Cdk1, and incubated with PLK-1 for pull-down experiments. The SPAT-1–PLK-1 complex was eluted with desthiobiotin (Elutions) and analyzed by SDS-PAGE. The membrane was blotted with PLK-1 (top) and SPAT-1 antibodies (bottom). (F) Western blot showing the result of the gel filtration analysis of Strep–SPAT-1 WT (top) and Strep–SPAT-1 13A (bottom) proteins. The proteins used to calibrate the column are indicated on the top.

Mentions: To identify the residues phosphorylated by CDK-1, we subjected SPAT-1, phosphorylated in vitro by human CyclinB/Cdk1, to tandem mass spectrometry (MS; liquid chromatography [LC]–MS/MS) analysis and identified 13 ((S/T)P) residues (S36, T57, T78, S119, S190, T229, S251, T328, S368, T456, T465, S504, and T518; Fig. 2, A and B; and Table S2). A nonphosphorylatable SPAT-113A mutant (with all S and T of the (S/T)P sites mutated to alanine) displayed a significantly dampened phosphorylation by CyclinB/Cdk1 (Fig. 2 C), indicating that these 13 residues are indeed the major CyclinB/Cdk1 phosphorylation sites. MS analysis of endogenous SPAT-1 or GFP::SPAT-1 immunoprecipitated from WT embryonic extracts confirmed that SPAT-1 is phosphorylated in vivo on at least 7 (S36, T57, S190, T229, S251, T328, and S504) out of the 13 residues that are phosphorylated by Cdk1 in vitro (Materials and methods; Fig. 2 B and Table S2).


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)

CyclinB/Cdk1 phosphorylates SPAT-1 at multiple sites. (A) Flow chart of the approach used to map SPAT-1 phosphorylation sites in vitro and in vivo. IP, immunoprecipitation. LC-MS/MS, tandem MS. (B) Table showing the amino acid position of the CDK-1–dependent phosphorylation sites in SPAT-1 protein sequence (F57C2.6b) with the 13 (S/T)p sites identified in vitro (highlighted in light gray), seven of which were confirmed in vivo (highlighted in dark gray). (C) Kinase assay conducted with purified Strep–SPAT-1 WT or Strep–SPAT-1 13A with human CyclinB/Cdk1 and γ-[32P]ATP. (top) SDS-PAGE gel stained with Coomassie shows protein loading. (bottom) Autoradiography indicates SPAT-1 phosphorylation status. (D) Strep pull-downs from insect Sf9 cells coexpressing PLK-1 and Strep–SPAT-1 WT (lanes 1 and 4), PLK-1 and SPAT-1 13A mutant (lanes 2 and 5), or PLK-1 alone (lanes 3 and 6) were analyzed by SDS-PAGE and probed with PLK-1 (top) and SPAT-1 (bottom) antibodies. A fraction (1/100) of the total protein extracts (Total) was loaded in the lanes 1–3. (E) Strep–SPAT-1 WT or the nonphosphorylatable 13A mutant version was immobilized on Strep-Tactin Sepharose beads, phosphorylated by CyclinB/Cdk1, and incubated with PLK-1 for pull-down experiments. The SPAT-1–PLK-1 complex was eluted with desthiobiotin (Elutions) and analyzed by SDS-PAGE. The membrane was blotted with PLK-1 (top) and SPAT-1 antibodies (bottom). (F) Western blot showing the result of the gel filtration analysis of Strep–SPAT-1 WT (top) and Strep–SPAT-1 13A (bottom) proteins. The proteins used to calibrate the column are indicated on the top.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: CyclinB/Cdk1 phosphorylates SPAT-1 at multiple sites. (A) Flow chart of the approach used to map SPAT-1 phosphorylation sites in vitro and in vivo. IP, immunoprecipitation. LC-MS/MS, tandem MS. (B) Table showing the amino acid position of the CDK-1–dependent phosphorylation sites in SPAT-1 protein sequence (F57C2.6b) with the 13 (S/T)p sites identified in vitro (highlighted in light gray), seven of which were confirmed in vivo (highlighted in dark gray). (C) Kinase assay conducted with purified Strep–SPAT-1 WT or Strep–SPAT-1 13A with human CyclinB/Cdk1 and γ-[32P]ATP. (top) SDS-PAGE gel stained with Coomassie shows protein loading. (bottom) Autoradiography indicates SPAT-1 phosphorylation status. (D) Strep pull-downs from insect Sf9 cells coexpressing PLK-1 and Strep–SPAT-1 WT (lanes 1 and 4), PLK-1 and SPAT-1 13A mutant (lanes 2 and 5), or PLK-1 alone (lanes 3 and 6) were analyzed by SDS-PAGE and probed with PLK-1 (top) and SPAT-1 (bottom) antibodies. A fraction (1/100) of the total protein extracts (Total) was loaded in the lanes 1–3. (E) Strep–SPAT-1 WT or the nonphosphorylatable 13A mutant version was immobilized on Strep-Tactin Sepharose beads, phosphorylated by CyclinB/Cdk1, and incubated with PLK-1 for pull-down experiments. The SPAT-1–PLK-1 complex was eluted with desthiobiotin (Elutions) and analyzed by SDS-PAGE. The membrane was blotted with PLK-1 (top) and SPAT-1 antibodies (bottom). (F) Western blot showing the result of the gel filtration analysis of Strep–SPAT-1 WT (top) and Strep–SPAT-1 13A (bottom) proteins. The proteins used to calibrate the column are indicated on the top.
Mentions: To identify the residues phosphorylated by CDK-1, we subjected SPAT-1, phosphorylated in vitro by human CyclinB/Cdk1, to tandem mass spectrometry (MS; liquid chromatography [LC]–MS/MS) analysis and identified 13 ((S/T)P) residues (S36, T57, T78, S119, S190, T229, S251, T328, S368, T456, T465, S504, and T518; Fig. 2, A and B; and Table S2). A nonphosphorylatable SPAT-113A mutant (with all S and T of the (S/T)P sites mutated to alanine) displayed a significantly dampened phosphorylation by CyclinB/Cdk1 (Fig. 2 C), indicating that these 13 residues are indeed the major CyclinB/Cdk1 phosphorylation sites. MS analysis of endogenous SPAT-1 or GFP::SPAT-1 immunoprecipitated from WT embryonic extracts confirmed that SPAT-1 is phosphorylated in vivo on at least 7 (S36, T57, S190, T229, S251, T328, and S504) out of the 13 residues that are phosphorylated by Cdk1 in vitro (Materials and methods; Fig. 2 B and Table S2).

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