Research ArticleMitosis

Aurora B opposes PP1 function in mitosis by phosphorylating the conserved PP1-binding RVxF motif in PP1 regulatory proteins

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Science Signaling  15 May 2018:
Vol. 11, Issue 530, eaai8669
DOI: 10.1126/scisignal.aai8669
  • Fig. 1 PP1 preferentially binds nonphosphorylated RV[S/T]F motifs in vitro.

    (A) Phosphorylated (P) or nonphosphorylated (nonP) versions of RV[S/T]F-containing peptides from the indicated proteins were spotted onto nitrocellulose membranes in the indicated amounts. The membranes were overlaid with a mixture of recombinant human PP1 and probed with a PP1-specific antibody. A peptide derived from the PP1-binding protein YLP motif–containing protein 1 (YLPM1) (RVRW) was used as a positive control, and the nonphosphorylable mutant version of this peptide (RARA) was used as the negative control. (B) Activated 5-carboxypentyl–Sepharose 4B N-hydroxysuccinimide ester (CH-Sepharose) beads were coupled to the phosphorylated or nonphosphorylated versions of the indicated RV[S/T]F peptides and incubated with clarified HeLa whole-cell extracts. After washing, proteins bound to the beads were eluted with SDS sample buffer, run on SDS–polyacrylamide gel electrophoresis (PAGE), and immunoblotted for PP1. The position of the 35-kDa mass marker is indicated. Peptides derived from the indicated proteins and sequences are shown in table S1. The data represent one of three independent experiments for both (A) and (B).

  • Fig. 2 RV[S/T]F motifs are phosphorylated in cells during mitosis.

    (A) HeLa cells were synchronized using thymidine-nocodazole (T/N) block and released in fresh media thereafter. Samples were collected at the indicated time points, immunoblotted for cyclin B1, and analyzed for phosphorylation within the RV[S/T]F motifs of the indicated proteins using in-house–generated phosphoepitope-specific antibodies. Async, asynchronized cells. Antibodies are characterized in fig. S2, and peptide sequences are shown in table S1. Samples were collected three times with the same synchronization technique, and the data represent one of three independent experiments. Equal loading was confirmed by immunoblotting for tubulin. Mass markers are indicated in kilodaltons. (B) Quantification of cells in each phase of the cell cycle at the indicated time points after synchronization as determined by FACS analysis.

  • Fig. 3 Global analysis of RV[S/T]F motif phosphorylation during mitosis.

    (A) The phosphoepitope-specific antibody generated using the peptide RRVpSFADK recognizes several proteins in immunoblots of HeLa cell lysates (top). Lysates from synchronized HeLa cells at the indicated times after release from T/N block were probed with this RVp[S/T]F (p-RV[S/T]F)–specific antibody to monitor changes in the phosphorylation status of this motif during the cell cycle. Phosphorylation of PP1 at Thr320 (p-Thr320) was also assessed at these time points. Equal loading was confirmed by immunoblotting for tubulin. Mass markers are indicated in kilodaltons. Three replicates of the p-RV[S/T]F immunoblot were quantified using ImageJ software and normalized to the mitotic (0.5 hours) sample (bottom). Error bars represent means ± SD. (B) Immunofluorescence staining of HeLa cells showing 4′,6-diamidino-2-phenylindole (DAPI), tubulin, and p-RV[S/T]F at different phases of the cell cycle. More than 50 cells were imaged for each condition in four independent experiments. Scale bar, 20 μm. a.u., arbitrary units.

  • Fig. 4 Effects of mitotic kinase inhibition on phosphorylation of PP1-binding RV[S/T]F motifs.

    (A) After mitotic arrest and treatment with the proteasome inhibitor MG132, HeLa cells were treated with the indicated kinase inhibitors The cells were harvested, and the lysates were separated by SDS-PAGE and immunoblotted for phosphorylated RV[S/T]F (p-RV[S/T]F, top). The blot was quantified using ImageJ software and normalized to the mitotic sample (bottom). MLN8054, Aurora A and Aurora B inhibitor; ZM447439 and hesperadin, Aurora B inhibitors; BI2536, PLK1 inhibitor; flavopiridol, CDK1 inhibitor. n = 3. **P < 0.01, paired Student’s t test. (B) To demonstrate the effectiveness and specificity of each kinase inhibitor, the same samples from (A) were immunoblotted to show the indicated phosphorylated proteins. H3 is phosphorylated on Ser10 by Aurora B; Aurora A is autophosphorylated on Thr288; Aurora B is autophosphorylated on Thr232; PP1 is phosphorylated on Thr320 by CDK1 (arrow); TCTP is phosphorylated on Ser46 by PLK1. n = 3. (C) HeLa cells were treated with inhibitors of Aurora B (hesperadin), Aurora A (Aurora A inhibitor I), CDK1 (roscovitine), or PLK1 (BI2536) for 2 hours before immunostaining for tubulin and phosphorylated RV[S/T]F (p-RV[S/T]F). Nuclei are indicated with DAPI. Control cells were not treated with any inhibitor. More than 50 cells were imaged for each condition in three independent experiments. Scale bar, 20 μm.

  • Fig. 5 Aurora B phosphorylates RV[S/T]F motifs during mitosis.

    (A) HeLa cells were arrested in mitosis with nocodazole, treated with either dimethyl sulfoxide (−) or the Aurora B inhibitor hesperadin (+) in the presence of the proteasome inhibitor MG132, harvested, and immunoblotted for phosphorylated RV[S/T]F motifs (p-RV[S/T]F). Mass markers are indicated in kilodaltons. (B and C) Immunofluorescence showing nuclei (DAPI), tubulin, and either H3 phosphorylated on Ser10 (p-H3S10, B) or p-RV[S/T]F (C) in mitotically arrested HeLa cells treated with the Aurora B inhibitor hesperadin. More than 15 cells were imaged for three independent replicates. (D) Difference in RVp[S/T]F staining was quantified for each condition, untreated (Control) and Aurora B inhibitor–treated (AurBi), by calculating the total cell fluorescence, using ImageJ. Error bars represent means ± SD. **P < 0.01, paired Student’s t test (n = 15). Scale bars, 20 μm.

  • Fig. 6 Phosphorylation of the RVxF motif by Aurora B inhibits PP1 binding.

    (A) Recombinant human PP1α/β/γ coupled to microcystin-Sepharose beads were used to test in vitro binding between PP1 and proteins from asynchronous (Async), mitotic, or Aurora B–inhibited (AurBi) HeLa extracts. After incubating the PP1-coupled beads with cell lysates, the bound proteins were eluted and tested for the presence of RIF1 by immunoblotting (top). Immunoblots were quantified using ImageJ software and normalized relative to RIF1 binding to PP1-coupled beads in extracts from asynchronous cells. Error bars represent means ± SD. *P < 0.05, paired Student’s t test (n = 3 independent experiments). (B) Nonphosphorylated and phosphorylated peptides containing the RV[S/T]F motifs from the indicated proteins coupled to activated CH-Sepharose beads were incubated with HeLa cell extracts. After washing, the bound proteins were eluted with SDS sample buffer and immunoblotted for PP1 (top). The sequences of the peptides are shown in table S1. Three replicates of the experiment were quantified and normalized relative to nonphosphorylated RIF1 peptide (bottom). Error bars represent means ± SD.

  • Fig. 7 Proposed model of PP1 regulation during the cell cycle.

    PPI is the catalytic subunit of the PPI holoenzyme, which includes both PP1 and any one of various regulatory proteins that bind to PP1 and control its catalytic activity. PP1 binds to RV[S/T]F motifs in the regulatory proteins during interphase. Upon activation of Aurora B during mitosis, these regulatory proteins are phosphorylated within the RV[S/T]F motifs. This phosphorylation leads to the dissociation of PP1 from the regulatory proteins thereby interfering with targeting of the phosphatase to its substrates. At the end of mitosis, these motifs are dephosphorylated, which regenerates the docking site for PP1, enabling the holoenzyme to target phosphorylated substrates for their dephosphorylation at mitotic exit.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/530/eaai8669/DC1

    Fig. S1. Consensus RV[S/T]F motif in PP1-interacting proteins.

    Fig. S2. Validation of phospho-specific antibodies by dot blots and immunofluorescence.

    Fig. S3. Validation and characterization of the RVp[S/T]F antibody.

    Fig. S4. Phosphorylation within RVS/TF motifs during mitosis.

    Table S1. List of peptide sequences used in this study.

    Table S2. List of proteins specifically bound to p-RV[S/T]F antibody and their binding behavior upon Aurora B inhibition.

    Table S3. Table containing SAINT analysis of the proteins enriched in the p-RV[S/T]F immunoprecipitations.

    Table S4. Table containing GO analysis based on biological processes for proteins enriched in p-RV[S/T]F immunoprecipitations.

    Table S5. Table containing GO analysis based on cellular component localization for proteins enriched in p-RV[S/T]F immunoprecipitations.

    Table S6. List of proteins containing “RV[S/T]F” motifs specifically enriched in the p-RV[S/T]F immunoprecipitation and their Aurora B sensitivity.

  • Supplementary Materials for:

    Aurora B opposes PP1 function in mitosis by phosphorylating the conserved PP1-binding RVxF motif in PP1 regulatory proteins

    Isha Nasa, Scott F. Rusin, Arminja N. Kettenbach,* Greg B. Moorhead*

    *Corresponding author. Email: moorhead{at}ucalgary.ca (G.B.M.); arminja.n.kettenbach{at}dartmouth.edu (A.N.K.)

    This PDF file includes:

    • Fig. S1. Consensus RV[S/T]F motif in PP1-interacting proteins.
    • Fig. S2. Validation of phospho-specific antibodies by dot blots and immunofluorescence.
    • Fig. S3. Validation and characterization of the RVp[S/T]F antibody.
    • Fig. S4. Phosphorylation within RVS/TF motifs during mitosis.
    • Legends for tables S1 to S6

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of peptide sequences used in this study.
    • Table S2 (Microsoft Excel format). List of proteins specifically bound to p-RV[S/T]F antibody and their binding behavior upon Aurora B inhibition.
    • Table S3 (Microsoft Excel format). Table containing SAINT analysis of the proteins enriched in the p-RV[S/T]F immunoprecipitations.
    • Table S4 (Microsoft Excel format). Table containing GO analysis based on biological processes for proteins enriched in p-RV[S/T]F immunoprecipitations.
    • Table S5 (Microsoft Excel format). Table containing GO analysis based on cellular component localization for proteins enriched in p-RV[S/T]F immunoprecipitations.
    • Table S6 (Microsoft Excel format). List of proteins containing "RV[S/T]F" motifs specifically enriched in the p-RV[S/T]F immunoprecipitation and their Aurora B sensitivity.

    [Download Tables S1 to S6]


    © 2018 American Association for the Advancement of Science

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