Research ArticleMitosis

Global assessment of its network dynamics reveals that the kinase Plk1 inhibits the phosphatase PP6 to promote Aurora A activity

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Science Signaling  15 May 2018:
Vol. 11, Issue 530, eaaq1441
DOI: 10.1126/scisignal.aaq1441
  • Fig. 1 Kinase activity–dependent Plk1 interactome.

    (A) Experimental design to identify kinase activity–dependent Plk1 interactors by SILAC. Heavy- and light-labeled HeLa cells were arrested in mitosis and treated with MG132. Heavy-labeled cells were treated with a specified kinase inhibitor or dimethyl sulfoxide (DMSO). Heavy- and light-labeled cells were mixed and lysed, and then, Plk1 and its interacting proteins were immunoprecipitated from the lysates, resolved by gel electrophoresis, and analyzed by LC-MS/MS. (B) Hierarchical clustering of quantitative differences in the binding of Plk1-interacting proteins in HeLa cells arrested with Taxol (−) or treated with Plk1 inhibitor (“P”; BI2536), Cdk inhibitor (“C”; flavopiridol), or staurosporine (“S”). (C) Comparison between quantitative MS data (heat map) with Western blot analyses of immunoprecipitated Plk1, the two stable interacting proteins kinesin family member 2C (KIF2C) and KIF14, the two CDK1 kinase activity–dependent Plk1-interacting proteins Astrin and p53 binding protein 1 (53BP1), and the two Plk1 kinase activity–dependent Plk1-interacting protein regulator of cytokinesis (Prc1) and mitotic kinesin-like protein 2 (Mklp2) upon differential kinase inhibitor treatment.

  • Fig. 2 Subcellular localization map of Plk1 interactors.

    Subcellular localization map of Plk1 interactors to kinetochores/centromeres, microtubule cytoskeleton and spindle poles, Golgi apparatus, nuclear envelope, endoplasmic reticulum, actin cytoskeleton, ribonucleoprotein (RNP) complexes, and ribosomes.

  • Fig. 3 Stable interactors of Plk1 include phosphorylation-dependent interactions.

    (A) Diagram depicting the distribution of kinase inhibitor–sensitive (yellow) and kinase inhibitor–insensitive (blue) Plk1 interactors and, below, their relative enrichment in candidate PBD motifs [Ser-Ser-Pro (SSP)/Ser-Thr-Pro (STP); dark gray] or not (light gray). (B) Experimental design to identify kinase activity–dependent Plk1 interactors using wild-type and Pincer mutant Plk1. HeLa cells expressing with Myc- or Flag-tagged wild-type or Pincer mutant Plk1 were arrested in mitosis and treated with MG132. Plk1 and its interacting proteins were immunoprecipitated, resolved by gel electrophoresis, and analyzed by LC-MS/MS. (C) Diagram depicting the distribution of kinase inhibitor–sensitive (yellow) and kinase inhibitor–insensitive (blue) Plk1 interactors and their ability to bind (white) or not bind (black) to Pincer mutant Plk1.

  • Fig. 4 Regulatory interaction of PP6 and Plk1.

    (A) Western blot (WB) analysis of Myc-tagged PP6 subunit binding to endogenous Plk1 in immunoprecipitates (IPs) from lysates of HeLa cells expressing the indicated Myc-tagged PP6 subunit. (B) Immunoprecipitation of Flag-tagged wild-type and Pincer mutant Plk1 from transfected HeLa cells and Western blot analysis of coprecipitated endogenous PP6R2. (C) Diagram depicting PP6R2 domain structure and location of PBD motifs. (D) Immunoprecipitations of wild-type, PBD1-mutant, and PBD2-mutant PP6R2 and Western blot analysis of coprecipitated Plk1. (E) Immunoprecipitation assessing the dependence of the PP6R2-Plk1 interaction on the activity of CDK1–cyclin B complex. Bacterially expressed and purified PP6R2 was immobilized on activated Sepharose and incubated with adenosine 5′-triphosphate (ATP) and CDK1–cyclin B complex or not, followed by addition to mitotically arrested (Taxol) HeLa cell lysate, isolation of PP6R2-containing complexes, and Western blotting for Plk1. (F) As described in (E), except that immobilized PP6R2 was incubated directly with mitotic HeLa cell lysates with or without additional ATP before isolation of PP6R2 complexes and Western blotting for Plk1. Bovine serum albumin (BSA)–Sepharose was used as a control (E and F).

  • Fig. 5 Regulation of PP6 phosphatase activity by Plk1 in vitro.

    (A and B) Western blotting (A) and quantification (B) of Aurora A (AurA) phosphorylation at Thr288 in mitotically arrested HeLa cells treated with increasing amounts of Plk1 inhibitor, BI2536. Lamin A/C was a loading control. (C) Followed by Western blotting for total and phosphorylated (Thr288) Aurora A after preincubation with PP6R2-PP6c and CDK1–cyclin B, Plk1, or both in in vitro kinase/phosphatase assays, as indicated (top). (D) Western blot analysis of wild-type (WT) and Plk1 phosphorylation site mutants of PP6R2 holoenzymes containing PP6c, in vitro. (E) Representative Western blots of total and phosphorylated (Thr288) Aurora A (E) under basal conditions in vitro (−) or upon incubation with wild-type or mutant PP6R2 holoenzyme described in (D). Blots (A to E) are representative (n = 3) of and quantified data (B, C, and E) are means ± SD of three biological replicates. *P < 0.05 and **P < 0.01.

  • Fig. 6 Regulation of PP6 phosphatase activity by Plk1 in cells.

    (A and B) Representative Western blot and analysis of total and phosphorylated (Thr288) Aurora A upon depletion of PP6c (A) or PP6R2 (B), Plk1 inhibition with BI2536, or both. Data are means ± SD of three biological replicates. *P < 0.05 and **P < 0.01.

  • Fig. 7 Model of regulatory feedback governing Aurora A, Plk1, and PP6.

    (A) In G2, Aurora A and Plk1 activities are low, and PP6R2-PP6c activity is high, serving as a check on premature Aurora A activation. (B) As cells enter mitosis, Aurora A is activated, and PP6R2 is primed by the CDK1–cyclin B complex, promoting Plk1 activation and phosphorylation-dependent inhibition of PP6. This results in a net increase in Aurora A phosphorylation and activity. (C) At the onset of anaphase, protein turnover of cyclin B, Plk1, and Aurora A breaks this feedback loop and resets the PP6R2-PP6c complex for the subsequent cell cycle.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/530/eaaq1441/DC1

    Fig. S1. SDS-PAGE gels of control and Plk1 IPs.

    Fig. S2. Comparison of the effects of flavopiridol and staurosporine on Plk1 interactions.

    Fig. S3. GO analyses.

    Fig. S4. Protein complexes in the Plk1 interactome.

    Fig. S5. First-degree neighbors of CDK1-targeted Plk1 interactors.

    Fig. S6. Regulatory subunit interactors of PP6 and Plk1.

    Fig. S7. Conservation of candidate PBD-binding motifs in PP6R2.

    Fig. S8. Termination of the Plk1-PP6 interaction.

    Fig. S9. Dependence of PP1c on Plk1 activity in dephosphorylating the Aurora A–TPX2 complex.

    Table S1. SILAC-based identification of kinase inhibitor–responsive Plk1 protein-protein interactions.

    Table S2. Number of SP/TP and SSP/STP motifs in Plk1 interactors.

    Table S3. Wild-type and Pincer mutant Plk1 protein-protein interactions.

    Table S4. SILAC-based identification of Plk1 inhibitor–responsive phosphorylation sites on PP6R2.

    Table S5. In vitro confirmation of CDK1–cyclin B phosphorylation occupancy of PP6R2.

  • Supplementary Materials for:

    Global assessment of its network dynamics reveals that the kinase Plk1 inhibits the phosphatase PP6 to promote Aurora A activity

    Arminja N. Kettenbach,* Kate A. Schlosser, Scott P. Lyons, Isha Nasa, Jiang Gui, Mark E. Adamo, Scott A. Gerber*

    *Corresponding author. Email: arminja.n.kettenbach{at}dartmouth.edu (A.N.K.); scott.a.gerber{at}dartmouth.edu (S.A.G.)

    This PDF file includes:

    • Fig. S1. SDS-PAGE gels of control and Plk1 IPs.
    • Fig. S2. Comparison of the effects of flavopiridol and staurosporine on Plk1 interactions.
    • Fig. S3. GO analyses.
    • Fig. S4. Protein complexes in the Plk1 interactome.
    • Fig. S5. First-degree neighbors of CDK1-targeted Plk1 interactors.
    • Fig. S6. Regulatory subunit interactors of PP6 and Plk1.
    • Fig. S7. Conservation of candidate PBD-binding motifs in PP6R2.
    • Fig. S8. Termination of the Plk1-PP6 interaction.
    • Fig. S9. Dependence of PP1c on Plk1 activity in dephosphorylating the Aurora Aâ€"TPX2 complex.
    • Legends for tables S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). SILAC-based identification of kinase inhibitor–responsive Plk1 protein-protein interactions.
    • Table S2 (Microsoft Excel format). Number of SP/TP and SSP/STP motifs in Plk1 interactors.
    • Table S3 (Microsoft Excel format). Wild-type and Pincer mutant Plk1 protein-protein interactions.
    • Table S4 (Microsoft Excel format). SILAC-based identification of Plk1 inhibitor–responsive phosphorylation sites on PP6R2.
    • Table S5 (Microsoft Excel format). In vitro confirmation of CDK1–cyclin B phosphorylation occupancy of PP6R2.

    [Download Tables S1 to S5]


    © 2018 American Association for the Advancement of Science

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