Bora Bridges Aurora-A Activation and Substrate Recognition of PLK1

  1. Jennifer A Miles
  2. Matthew Batchelor
  3. Martin Walko
  4. Vanda Gunning
  5. Andrew J Wilson
  6. Megan H Wright
  7. Richard Bayliss

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Abstract

Cell cycle transitions are orchestrated by regulated protein kinase activities. The activation of PLK1 in late G2 is a decisive step for cells to enter mitosis, requiring phosphorylation of its activation loop by Aurora-A, facilitated by the intrinsically disordered protein Bora. Despite its biological importance, the structural basis of this mechanism has remained unresolved. Here, we present models of the Aurora-A/Bora complex and the ternary Aurora-A/Bora/PLK1 complex, validated with site-specific mutagenesis, biochemical assays and NMR spectroscopy. Bora wraps around the N-lobe of Aurora-A, occupying the pockets used by its other activators. A CDK1 phosphorylation site on Bora (Ser112) mimics the structural role of Aurora-A activation loop phosphorylation within a TPX2-like binding motif. In the ternary complex, Bora bridges the two kinases, orienting the activation loop of PLK1 towards the active site of Aurora-A. Bora residues 56-66 form a critical interface with a conserved pocket on the PLK1 C-helix that is analogous to the TPX2-binding Y-pocket of Aurora-A. Aurora-A phosphorylation of Bora Ser59 creates an additional interaction that increases the efficiency of PLK1 phosphorylation. These findings deepen our understanding of how Aurora-A activity is fine-tuned by its disordered binding partners and establish a mechanistic framework for its Bora-dependent activation of PLK1.

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  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    We appreciated the positive, detailed and helpful feedback from all three reviewers.

    Reviewer 1.

    Minor comments.

    1. In the introduction, on page 2, the authors seem a little confused about the Plk1 Polo-box domain - text as written: "...kinase domain linked to tandem Polo-box domains (PBD)", and cite a review paper. Actually, there is only a single Polo-box domain in these kinases, which contains both Polo-boxes and a bit of the upstream linker region. The "PBD" terminology denotes his 2-Polo-box +linker structure. Perhaps it would be better here to cite the PBD structure (Elia et al., Cell, 2002) as a primary citation here.

    Response: Thank you for finding this error, the text has been updated and the new citation included within the text on line 65.

    1. Similarly, the line "...during the G2/M transition following successful DNA damage repair" cites the Seki et al paper, but those findings are shown in the Macurek et al paper, not the Seki et al paper.

    ____Response: Thank you for finding this error, the new citation included within the text on line 69.

    1. Using the model of the ternary complex as shown in Figure 1B, deletion constructs of Bora missing regions within the disordered loops, but still retaining the residues that bind the PBD, FW pocket and Aurora A, can be modeled and tested to see if such deletions can improve the ipTM scores and binding affinity.

    Response: ____AlphaFold3 modelling was attempted with shorter regions of Bora to see the effect on the ipTM scores. Unfortunately, when Bora was reduced to shorter sequences, such as 18-88 or 18-45 modelled with 68-120, the models became inconsistent and of a low quality. Models were also created including the short region of Bora surrounding Ser252 that interacts with the polo box domain as well as Bora 18-120, but this had minimal effect on the calculated iPTM scores.

    1. On page 5, "S112A" within the sentence "Unexpectedly, the F56A/W58A Bora was less efficiently phosphorylated on S112A (Supplementary Figure S11, F compared to H and Supplementary Table S4)." This should be "S112".

    Response: ____Thank you for spotting this, the error has been corrected.

    1. In the assays shown in Figure 2D, the presence of excess F56AW58A Bora that remained unphosphorylated on S112 may complicate the interpretation of the results. Can the authors show that the S112-phosphorylated F56AW68A Bora is predominantly bound to Aurora A in such a mixture, perhaps by NMR using labelled pS112 F56AW58A Bora and unlabeled S112 F56AW58A Bora?

    ____Response: 15N13C labelled of Bora 18-120 F56A W58A was produced and assigned. We then phosphorylated a sample using ERK2, tracking with NMR, and when the reaction had progressed to a 50:50 mixture of pSer112 and Ser112 (based on peak intensities) the kinase activity was quenched by addition of EDTA to sequester Mg2+. This produced a solution containing both pS112 and unphosphorylated S112 Bora species with marker peaks in HSQC spectra that could be used to directly compare Aurora-binding to the two species. Aurora-A was introduced to the sample and the peak intensities were monitored. Although both species are affected, there is much greater peak loss from the pS112 related peaks than those for unphosphorylated S112. This indicates that Aurora-A still preferentially binds pS112 Bora over S112 Bora when the F56A W58A mutation is present. This data has been included in Supplementary Figure S11.

    1. Please expand Figure 3A to better show the FW pocket-forming residues on Plk1.

    Response: ____Figure 3 has been amended to reduce the size of the sequence alignments so that 3A could be made slightly larger.

    1. It would be helpful to label the peaks in the mass spectra in Fig. S11 with the phospho-species that they correspond to.

    Response: ____This information has been added to the mass spectra in Fig. S11 (now supplementary Figure S14) to make them easier to view.

    1. In the last paragraph on page 7, "see we" in the sentence "As well as a decrease in intensity around pSer112 in Bora, see we an overall effect with decreased intensity across most of the Bora sequence." Should be corrected to "we see".

    Response: ____Thank you for spotting this, the error has been corrected.

    1. While not required, it would be helpful if binding or Bora to Aurora A after Erk2 phosphorylation could be shown using fluorescence polarization or ITC to lend additional support to the NMR data for S112 and S59 phosphorylation and for CEP192 and TPX2 competition.

    Response: ____This question has been partially answered in previous work by Tavernier et al. (2021), who showed improved binding of Aurora-A to Bora after Erk phosphorylation (by SPR), and they used labelled-TPX2 for a series of competition FP assays in that and the recent parallel study (Pillan et al. 2025).

    We made initial efforts to perform additional FP assays using longer sections of Bora with different phosphorylation states but without success (perhaps due to the multisite-binding nature of the Bora–Aurora interaction, and difficulties with directly expressing phosphorylated Bora). The revised manuscript now includes some additional NMR data to show improved Bora–Aurora-A interaction after phosphorylation at Ser59 (Supplementary Figure S12).

    1. The Aurora A phosphorylation motif has been further defined beyond that reported by the Pinna lab in 2005. Notably, the Ser-59 sequence on Bora (F-R-W-S-I), has, in addition to dominant selection for AR in the -2 position, both favorable -1 (W) and +1 (I) positions based on peptide library measurements (Alexander et al., Science Signaling 2011), further arguing that it may be an excellent Aurora A phosphorylation site.

    Response: ____Thank you for highlighting this publication and how it further reinforces the likelihood of Ser59 being an effective substrate for Aurora-A, this should have been included in the original manuscript. This citation has now been included.

    1. Have the authors tried to model the Drosophila melanogaster Aurora A-Bora-Polo complex to see if the Asn substitution of Bora Ser59, and the expected loss of the interactions between Bora pSer59 and Plk1 Arg59 and Aurora A Arg205 are compensated by other features?

    Response: ____A ternary complex between the Drosophila melanogaster orthologues was modelled using AlphaFold3 (Uniprot code PLK1 (Q9VVR2 72-165), Aurora-A kinase (Q9VGF9) 151-411 and PLK1 (P52304 21-280)). This model was analysed using PDBe PISA to identify potential interactions between the three proteins, focusing on residues that are not conserved between the human and Drosophila sequences. From this model a potential salt bridge was identified between Drosophila Bora Lys120 and PLK1 Glu93 that would not occur in the human ternary complex given Lys120 is replaced with an asparagine. This could be an alternative (kinase-independent) method for improved Bora-PLK1 interaction. When comparing the Bora:Aurora-A side of the predicted interface and focusing on the short region of Bora in between Aurora-A and PLK1, there were no clear differences seen in the residues predicted to bind to Aurora-A. This modelling has been included in Supplementary Figure S10 C and D.

    1. Given the relevance of the recent publication from Zhu et al. to this study, the authors may want to comment on, or test, the relative importance of PKA and Aurora A as a potential kinase for Bora S59. While those authors argue that PKA phosphorylates Bora on Ser-59, one could easily imagine a model in which either PKA or Aurora A could initially phosphorylate that site followed by a propagation step after initial Aurora A activation, in which Aurora A phosphorylation of Bora Ser-59 is the dominant process.

    Response: ____A brief discussion of this recent publication has been added to the discussion, highlighting the similarities between the two publications and the importance of pSer59, as well as suggesting that in cellulo this modification could be achieved via more than one pathway. We also include some additional NMR data to show improved Bora–Aurora-A interaction after phosphorylation at Ser59 (Supplementary Figure S12).

    Reviewer 2.

    Minor comments.

    Page 5: '... a K82R PLK1 mutant was used to increase the stability of the protein' - It is not clear how this mutation confers increased stability of the protein. The authors do not show any data to support this. Isn't the PLK1 K82R an ATP-binding-deficient, kinase-inactive mutant?

    Response: ____Thank you for spotting this, the text has been updated to clarify that this version of PLK1 was used as it is acting as a substrate in the in vitro assay as we didn’t want to see any PLK1 activity within this assay.

    All panels showing the Alphabridge diagram - it would be helpful if pictorial definitions of the colour codes were provided with corresponding score ranges (in addition to the description in the figure legend).

    Response:____The AlphaBridge images have been updated to include details about the plDDT scores each of the different colours refer to.

    Fig 2B - The Fluorescence anisotropy assay curves do not reach a plateau. Though the effect of mutation on binding affinity is pretty clear, if possible, I suggest including more data points at higher concentrations and estimating apparent Kd values.

    __Response:____The direct binding assay was repeated with a higher concentration of PLK1 in order to try and see a top plateau. This was successful and has been included in Figure 2B (shown in black). The measured Kd was 24 ± 3 µM. __

    The cartoon representation of the structures and molecular interfaces - better to avoid shadows, as they compromise the clarity of the figures, particularly the ones where side chains are shown in stick representation.

    Response:____The structural images have been remade to remove the shadows and improve the clarity of the images.

    It is important to discuss how the parallel studies by Verza et al. and Pillan et al. complement this study, highlighting similarities and differences.

    Response:____References to these two publications and details on the similarities and differences seen are now included in the discussion.

    Reviewer 3.

    Major comments

    It would be helpful to measure the level of pThr210 PLK1 in some experiments and graph the data. The current presentation is Fig. 2D-E is qualitative rather than quantitative.

    Response:____Graphs displaying the levels of pThr210 produced in the assay are now shown in Supplementary Figure S4.

    Have the authors measured the binding affinity of the F/W mutant Bora for PLK1 using the assay in Fig. 2B? Likewise, for Fig. 7 the S59 mutant could be tested to see if it affects PLK1 binding or activation.

    Response:____The direct binding assay has been repeated with the use of a FAM-Bora peptide that incorporates the F56A W58A mutation which shows reduced binding (Figure 2B, shown in blue). A version of the Bora peptide phosphorylated on Ser59 was also tested in the direct binding assay and this shows a similar affinity for PLK1 to the wild-type sequence (Figure 2B, shown in red compared to the wild-type shown in black).

    It would be helpful if measurements of pThr210 PLK1 for all conditions were shown in the graph Fig. 7F.

    Response:____This graph has been updated to include the levels of phosphorylation seen for PLK1 in all of the conditions tested.

    Minor comments

    I found Figure S1B easier to understand than Fig S1A and Fig 1A-B. Some of the supplemental data Fig. S1C-E could be moved to a revised Figure 1, dropping the current Fig. 1A-B. Can the interaction plots (Fig. S1C-D) be rotated to have the same original at the top and order of proteins (i.e. Bora > Aurora A > {plus minus} PLK1 depending on the plot).

    Response:____Figure 1 and S1 have been rearranged to hopefully make them easier to understand, with all AlphaFold3 models of the full-length sequences kept in the supplementary figure and the focus in 1B just on the truncated model. The AlphaBridge plots have been rotated as suggested.

    Figure 3F. Typo "Strongyl" not "Strongly".

    Response:____Thank you for spotting this, this has been corrected in the updated manuscript.

    Figure 3 could be supplemental material.

    Response:______Thank you for your suggestion, but we have decided to keep this as a main figure.__

    Fig. 7E. Run a positive control reaction +ERK2 on the second gel to allow direct comparison of pThr210 across all the conditions tested.

    Response:____These samples have been rerun on the same membrane and the levels of phosphorylation have been quantified and included in Figure 7F.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary.

    Miles and co-workers have carried out a careful and high-quality study of the activation mechanisms of the mitotic kinase PLK1. Multiple proteins have been implicated in PLK1 activation and localisation as cell enter and pass through mitosis. Initial activation of PLK1 is promoted by a complex of Bora with another kinase Aurora A. Later in mitosis, this activated PLK1 associates with mitotic spindle and centrosome proteins regulating different aspects of mitosis and cytokinesis. In this study, Miles et al. extend previous work on this question by proposing and testing detailed models for Bora/Aurora A-mediated activation of PLK1 to elucidate the mechanism of this reaction.

    Using the latest Alphafold they generate a series of models of the PLK1/Bora/Aurora A complex to home in on the key regions mediating interactions of the three proteins. This approach suggests an arrangement where the first ~120 amino acids of Bora wrap Aurora A and create an interaction surface for the N-terminal kinase domain of PLK1. This orients Thr210 in PLK1 towards Aurora A creating a situation likely favourable for phosphorylation, although has the authors discuss there are some caveats to this. A further prediction of the modelling helps explain the requirement for Bora phosphorylation to promote the interaction with Aurora A. This data is presented in Fig. 1 and Fig. S1-S3.

    In the subsequent figures the details of this model are tested using biochemical assays and structural biology methods to validate key predictions. First the PLK1 interaction with Bora was shown to require the conserved F/W motif of Bora and a conserved pocket close to R106 on PLK1 (Fig. 2 and 3). In reconstituted PLK1 activation assays the F/W motif mutant Bora showed greatly attenuated pThr210 phosphorylation. This reaction also required phosphorylation of Bora at S112, presumably due to the interaction with Aurora A. An R106A mutant PLK1 showed reduced binding to Bora and reduced kinase activation. This data is clear and provides compelling support for the model.

    Using NMR the authors then investigate the interaction between Bora and Aurora A, and more specifically the requirement for Bora phosphorylation at Ser112. The NMR data in Fig. 4 and Fig. 6 provide good support for the Alphafold model. A helpful comparison with known Aurora A binding proteins is also shown to highlight the way CEP192, TPX2 and TACC3 contact a series of conserved pockets on the surface of Aurora A which are common to the Bora interaction. S59 phosphorylation by Aurora A is also shown to play an important role in contacting PLK1 and is required for pThr210 phosphorylation.

    In summary, the authors have made valuable progress in working out details of the PLK1 activation mechanism, that extends previous work in the field.

    Major comments.

    It would be helpful to measure the level of pThr210 PLK1 in some experiments and graph the data. The current presentation is Fig. 2D-E is qualitative rather than quantitative.

    Have the authors measured the binding affinity of the F/W mutant Bora for PLK1 using the assay in Fig. 2B? Likewise, for Fig. 7 the S59 mutant could be tested to see if it affects PLK1 binding or activation.

    It would be helpful if measurements of pThr210 PLK1 for all conditions were shown in the graph Fig. 7F.

    Minor comments.

    I found Figure S1B easier to understand than Fig S1A and Fig 1A-B. Some of the supplemental data Fig. S1C-E could be moved to a revised Figure 1, dropping the current Fig. 1A-B. Can the interaction plots (Fig. S1C-D) be rotated to have the same original at the top and order of proteins (i.e. Bora > Aurora A > {plus minus} PLK1 depending on the plot). Figure 3F. Typo "Strongyl" not "Strongly". Figure 3 could be supplemental material. Fig. 7E. Run a positive control reaction +ERK2 on the second gel to allow direct comparison of pThr210 across all the conditions tested.

    Significance

    Timely and orchestrated activation of multiple mitotic protein kinases is crucial for the alignment and segregation of chromosomes, and for the process of cell division. In this study the authors explore how activation of the mitotic kinase PLK1 is triggered by another mitotic kinase Aurora A, and the role played by a scaffold protein Bora.

    Strengths: Detailed analysis of mechanism using biochemical and structural approaches.

    Limitations: The study is focussed on the biochemical and structural mechanisms rather than the cellular outcomes. Some data would benefit from additional quantitative measurement.

    Relevance: Cancer and cell biology due to the role of Aurora A in many cancers.

    Reviewer expertise: Biochemistry, molecular and cell biology.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    PLK1 is one of the master regulators of cell division. The activation of PLK1 requires the activation loop phosphorylation at T210, mediated by Aurora A kinase. However, Aurora A phosphorylation of PLK1 T210 requires Bora, one of the several activators of Aurora A kinase. While the molecular requirement of Aurora A kinase and Bora for PLK1 activation is well established, the mechanistic understanding of how Bora facilitates PLK1 activation by Aurora A has remained an important open question for a long time. Exploiting the latest development in AI-driven structure prediction, three independent studies provide a structural and mechanistic basis for PLK1 activation by Aurora A and Bora. Here, Miles et al. have generated AlphaFold models, further characterised some of the interfaces using NMR, and validated the contribution of intermolecular interactions at suggested interfaces in vitro using recombinant proteins in kinase assays. Overall, this is a well-executed work providing important new insights into our understanding of the activation of the critical regulator of cell division, PLK1. However, as the authors have highlighted in the discussion section, one limitation of this modelling study is that the models still do not entirely explain how these interactions facilitate the phosphorylation of Thr210ur, as this residue is oriented far away from Aurora A's active site for the reaction to take place. Despite this limitation, I believe this is an important work that advances our understanding significantly.

    Comments:

    Experimental data satisfactorily support claims. Hence, most of my comments are minor in nature.

    Points to consider during revision:

    Page 5: '... a K82R PLK1 mutant was used to increase the stability of the protein' - It is not clear how this mutation confers increased stability of the protein. The authors do not show any data to support this. Isn't the PLK1 K82R an ATP-binding-deficient, kinase-inactive mutant?

    All panels showing the Alphabridge diagram - it would be helpful if pictorial definitions of the colour codes were provided with corresponding score ranges (in addition to the description in the figure legend).

    Fig 2B - The Fluorescence anisotropy assay curves do not reach a plateau. Though the effect of mutation on binding affinity is pretty clear, if possible, I suggest including more data points at higher concentrations and estimating apparent Kd values.

    The cartoon representation of the structures and molecular interfaces - better to avoid shadows, as they compromise the clarity of the figures, particularly the ones where side chains are shown in stick representation.

    It is important to discuss how the parallel studies by Verza et al. and Pillan et al. complement this study, highlighting similarities and differences.

    Significance

    As highlighted in the summary, a mechanistic understanding of how PLK1 is activated by Aurora A kinase and its activator Bora has remained a long-standing open question. As PLk1 is one of the major regulators of cell division, which exerts its function (via phosphorylating numerous substrates) during different stages of mitosis, understanding its activation mechanism is of critical interest for those working on the cell cycle in general and cell division in particular. A key limitation of this study is the lack of any cellular functional evaluation of the interaction interfaces.

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    Referee #1

    Evidence, reproducibility and clarity

    Miles et al. used a combination of AlphaFold modeling, biochemical assays of mutant constructs and NMR spectroscopy to model the ternary complex of Aurora A, Bora and Plk1, and elucidate how Bora can act as a molecular bridge that facilitates the phosphorylation of the activation loop Thr210 within Plk1 by Aurora A. Their studies identified an interaction between residues 52-73 within Bora and the 'FW' pocket on the N-terminal lobe of Plk1, which binds Phe56 and Trp58 of Bora. Additionally, Ser59 of Bora was identified as a good Aurora A substrate using a Bora peptide array, and pSer59 was predicted to form bridging interactions with Aurora Arg205 and Plk1 Arg59. This was supported by NMR and biochemical assays. In addition, the authors validate that phosphorylation of Ser-112 on Bora enhances stabilization of the Aurora A-Bora complex Overall, the model revealed novel details of the interactions within the Aurora A-Bora-Plk1 ternary complex that are supported by the biochemical and NMR data. The work will be of significant interest to basic scientists whose work involves protein kinase signaling, cell division/mitosis, signal transduction, and cancer biology. We recommend publication of this manuscript with the following minor changes and additions.

    1. In the introduction, on page 2, the authors seem a little confused about the Plk1 Polo-box domain - text as written: "...kinase domain linked to tandem Polo-box domains (PBD)", and cite a review paper. Actually, there is only a single Polo-box domain in these kinases, which contains both Polo-boxes and a bit of the upstream linker region. The "PBD" terminology denotes his 2-Polo-box +linker structure. Perhaps it would be better here to cite the PBD structure (Elia et al., Cell, 2002) as a primary citation here.
    2. Similarly, the line "...during the G2/M transition following successful DNA damage repair" cites the Seki et al paper, but those findings are shown in the Macurek et al paper, not the Seki et al paper.
    3. Using the model of the ternary complex as shown in Figure 1B, deletion constructs of Bora missing regions within the disordered loops, but still retaining the residues that bind the PBD, FW pocket and Aurora A, can be modeled and tested to see if such deletions can improve the ipTM scores and binding affinity.
    4. On page 5, "S112A" within the sentence "Unexpectedly, the F56A/W58A Bora was less efficiently phosphorylated on S112A (Supplementary Figure S11, F compared to H and Supplementary Table S4)." This should be "S112".
    5. In the assays shown in Figure 2D, the presence of excess F56AW58A Bora that remained unphosphorylated on S112 may complicate the interpretation of the results. Can the authors show that the S112-phosphorylated F56AW68A Bora is predominantly bound to Aurora A in such a mixture, perhaps by NMR using labelled pS112 F56AW58A Bora and unlabeled S112 F56AW58A Bora?
    6. Please expand Figure 3A to better show the FW pocket-forming residues on Plk1.
    7. It would be helpful to label the peaks in the mass spectra in Fig. S11 with the phospho-species that they correspond to.
    8. In the last paragraph on page 7, "see we" in the sentence "As well as a decrease in intensity around pSer112 in Bora, see we an overall effect with decreased intensity across most of the Bora sequence." Should be corrected to "we see".
    9. While not required, it would be helpful if binding or Bora to Aurora A after Erk2 phosphorylation could be shown using fluorescence polarization or ITC to lend additional support to the NMR data for S112 and S59 phosphorylation and for CEP192 and TPX2 competition.
    10. The Aurora A phosphorylation motif has been further defined beyond that reported by the Pinna lab in 2005. Notably, the Ser-59 sequence on Bora (F-R-W-S-I), has, in addition to dominant selection for AR in the -2 position, both favorable -1 (W) and +1 (I) positions based on peptide library measurements (Alexander et al., Science Signaling 2011), further arguing that it may be an excellent Aurora A phosphorylation site.
    11. Have the authors tried to model the Drosophila melanogaster Aurora A-Bora-Polo complex to see if the Asn substitution of Bora Ser59, and the expected loss of the interactions between Bora pSer59 and Plk1 Arg59 and Aurora A Arg205 are compensated by other features?
    12. Given the relevance of the recent publication from Zhu et al. in https://doi.org/10.1038/s41467-025-63352-y to this study, the authors may want to comment on, or test, the relative importance of PKA and Aurora A as a potential kinase for Bora S59. While those authors argue that PKA phosphorylates Bora on Ser-59, one could easily imagine a model in which either PKA or Aurora A could initially phosphorylate that site followed by a propagation step after initial Aurora A activation, in which Aurora A phosphorylation of Bora Ser-59 is the dominant process.

    -Dan Lim and Michael Yaffe

    Significance

    The work is well done and clearly presented.

  5. Version published to 10.1101/2025.08.08.668882 on bioRxiv