A bifunctional kinase–phosphatase module balances mitotic checkpoint strength and kinetochore–microtubule attachment stability

  1. Andrea Corno
  2. Marilia H Cordeiro
  3. Lindsey A Allan
  4. Qian‐Wei Lim
  5. Elena Harrington
  6. Richard J Smith
  7. Adrian T Saurin

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Abstract

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore–microtubule error‐correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase–phosphatase pair (PLK1‐PP2A) that engage in negative feedback from adjacent phospho‐binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1‐MELT motifs, implicating PLK1‐PP2A in controlling auto‐amplification of MELT phosphorylation. In support, KNL1‐BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB‐PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore–microtubule attachments, induces chromosome segregation defects and prevents KNL1‐BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

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  1. Version published to 10.15252/embj.2022112630
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    Reply to the reviewers

    General Comments

    We thank all reviewers for providing very detailed, knowledgeable, and informative reviews.

    All reviewers were complementary about the data and the rigor of the study. Reviewers 2 and 3 commented on the significance of the work, and their assessments were complementary, specifically about the fact that it bridges several previous studies and links these to kinase-phosphatase regulation on the BUB complex. We agree that this a major strength of the work. That is why we also believe the comment by reviewer 1 that “most of the phenotypes/observations are consistent with the literature and not surprising” is actually a strength and not a weakness. Sometimes manuscripts that bring together various different findings into one conceptual model can be very powerful, even if each finding in isolation is not so surprising. In this case, the concept that a dual kinase-phosphatase module integrates two major mitotic processes will, we believe, prove to be a significant breakthrough that helps to explain how these processes are properly integrated at kinetochores.

    The main criticism of all reviewers related to the interpretations and writing style, which in general, we felt were valid. We will take on board all these comments, reword the manuscript during revision, and provide a detailed response to each of these points at resubmission.

    In terms of points requiring new experiments, there were 3 in total:

    1. Reviewers 1 and 3 raised an important issue about the feedback loop which will be addressed with new experiments to uncouple the feedback.

    2. Reviewer 2 made an important point about KNL1 levels, including a good suggestion to perform FRAP analysis to examine BUB complex dynamics when MELT numbers are increased. We will carry out this experiment prior to revision.

    3. Reviewer 1 had a second major comment regarding the modulation of MELT number and how this cannot be directly linked to PLK1/PP2A levels. We have 3 new experiments to add regarding this, performed already, which are discussed in the section below.

    All other comments were textual points that in most case we felt were valid. They showed that all reviewers had a very good grasp of the paper, the concepts, and the field in general. So, we finish by thanking all reviewers again for their thorough and detailed assessments of our manuscript. The comments they raised will help us to improve the manuscript after revision.

    Description of the planned revisions

    Three main points:

    1) The role of the feedback loop [reviewers 1 and 3]:

    The general issue is explained succinctly by reviewer 3’s comment:

    “The argument linking the negative feedback loop to biological functions is not straightforward. The authors provide evidence in Figure 1 for regulatory pathways between docked PLK1 and bound PP2A. However, their assays in Figure 2 bypass the feedback loop by directly modulating PP2A activity. These experiment supports an argument that the kinase/phosphatase activity balance is important, but do not address the feedback loop specifically (which could potentially be done using mutations that disrupt the feedback regulation). The claim that "a homeostatic feedback loop maintains an optimal balance of PLK1 and PP2A on the BUB complex" is too strong because there is no direct evidence connecting the feedback loop to optimal function.”

    This is a good point that we will address at revision. We demonstrate that the enzymes regulate each other on the BUB complex in figure 1 (PLK1 recruits PP2A, and PP2A removes PLK1), which balances their levels on the BUB complex. To determine consequences of upsetting this balance we locked either the kinase-bound or phosphatase-bound states (Figure 2). Importantly, this is required to assess direct phenotypes associated with each, but it does not directly demonstrate the role of the feedback loop. To do this we will generate mutants, as suggested, and analyse their phenotypes.

    We will mutate the PLK1 binding site (T620A) and recover the PLK1-regulated sites in the KARD motif to phospho-mimicking aspartates (S676D/T680D), analyzing effects on PLK1/PP2A recruitment, chromosome alignment and SAC strength. We predict that this will remove PLK1 and recover some PP2A, but to lower levels overall than the BUBR1-B56 fusion. In that case the phenotypes will probably be milder, but that would not change the overall conclusions.

    We maintain that locking PLK1 on its phospho-binding site (in BUBR1-DPP2A) is the ideal scenario to test direct PLK1 roles, but we will also now create alanine mutants of the PLK1 site (S676A/T680A) and the CDK1 site (S670A) to address the feedback loops controlled by CDK1 and PLK1. Our prediction is that these will skew the balance towards PLK1, without fully removing PP2A, again likely to produce milder intermediate phenotypes.

    It is definitely worth testing these predictions, because it would directly address the role of the feedback loop and it would avoid relying solely on “artificially high levels” as mentioned by reviewer 1. One final point on this however, the PLK1 recruitment in DPP2A cells is not artificial – it is PLK1 bound to its native phospho-motif when PP2A is unbound (without any feedback from PP2A this phospho-site and PLK1 binding increase to the observed maximal levels). The fusion of B56 is admittedly less optimal, but this does still lock the phosphatase-bound state in a set stoichiometry, crucially in the absence of kinase. This is required to assess direct phosphatase effects. These PLK1/PP2A levels may well be higher than observed physiologically on the BUB complex when considering the behavior of all BUBR1 molecules, since we doubt they ever reach 1:1 stoichiometry with either PLK1 or PP2A. However, the feedback loop is operating within individual molecules (figure 1), which may well individually flip between PLK1 or PP2A bound states. This may occur on certain molecules at specific times. Therefore, locking the PLK1.PP2A-bound state on all molecules is, in our opinion, still a valid and useful perturbation to assess function of these two states.

    2) The increase recruitment of BUB1-PLK1/PP2A when MELT numbers are increased [reviewer 2]

    "While in the 12x and 19x mutant conditions there are more molecules of BUBs per Knl1, the overall BUB levels are the same as in wild-type controls. Since the MELT repeat used throughout the paper is a consensus sequence that is likely optimal for BUB binding, it is possible that the phenotypes of the 12x and 19x mutants are explained because of an increase in the affinity of BUBs for Knl1 rather than overall levels. This would also help explain why Knl1 and BUBs are observed at the spindle midzone in the 19x mutant (Fig. S4)"

    The reviewer raises an important issue here, when stating that increasing MELT numbers decreases KNL1 kinetochore recruitment. This has the net effect of normalizing overall BUB1-PLK1/PP2A kinetochore levels, even though BUB1-PLK1/PP2A recruitment per KNL1 molecule is increased. That is why we were careful to state BUB1-PLK1/PP2A were increased “on each KNL1 molecule” and not “on kinetochores” when referring to the effect in the 12x/19x MELT mutant. However, this could easily be misinterpreted so this point will be clarified at revision.

    The issue of why the phenotype is so dependent on kinase/phosphatase level per KNL1 molecules is an important one however, which has puzzled us until now. We think the suggestion to look at turnover by FRAP is a good one, because enhanced binding strength could underlie the phenotype here, and potentially explain the lack of disassembly at anaphase. We will perform these experiments at revision to see if they can clarify the issue.

    3) The link between MELT number and PLK1/PP2A levels [Reviewer 1]

    “My second comment relates to the fact that the two parts of the paper are not directly linked although the authors try to do this. They nicely manipulate the MELT repeats on KNL1 to change the number of Bub complexes. However, they cannot directly link the data to changes in Plk1 and PP2A-B56 levels only as many other things are changing. By increasing MELT numbers Bub complex and Mad1/Mad2 levels increase as well as an example and this makes interpretations complicated. To me these experiments are not addressing the main conclusions of the paper.”

    We do not agree with this overall assessment, but there are two elements to this comment: the effect of modulating MELT number on SAC strength (and its link to PLK1) or on KT-MT stability (and link to P2A). We will therefore discuss each separately:

    For SAC regulation, we feel that the data is clear and the interpretations are justified, although we will add new data to support this point after revision. Increasing MELT number causes defects in MELT-BUB dissociation and SAC silencing (4a-c). Importantly, these phenotypes can be completely rescued by inhibiting PLK1 (4d-e). So, we do link the effects of high MELT number to PLK1 activity. Our interpretation is that when MELT numbers are increased the ability of PLK1 to phosphorylate these motifs and maintain the SAC platform is enhanced (when MPS1 is inhibited pharmacologically or upon KT-MT attachment). So, whilst it is true that many factors, such as the kinetochore levels of BUB/MAD1/MAD2, are crucial for the SAC, the ability of PLK1 to maintain these levels (via pMELT-BUB1) is crucial and that changes as MELT number increases. This contributes directly to the observe SAC silencing phenotype, as confirmed by the complete rescue of this phenotype after PLK1 inhibition.

    We did also explore the possibility that increased BUB1 activity could also contribute to SAC strengthening, for example, by enhancing Aurora B recruitment to centromeres. However, BUB1 inhibition did not alter SAC strength or MELT dephosphorylation kinetics. We will add this data after revision.

    We also evaluated the levels of phosphorylated MAD1-pT716, which is important for MCC assembly (Ji et al. 2017, Ji et al. 2018, Faesen, 2017). Our data show that WT and 19xMELT exhibit similar MAD1-pT716 levels during a nocodazole arrest and following MPS1 inhibition. In summary, the main changes we observe are elevated BUB1 levels due to MELT phosphorylation, and increased BUB1 phosphorylation on pT461 (as shown in Figure 4h). All this points towards a localized effect of PLK1 on/around the BUB complex. We will add this data and make this point clearly at revision.

    For KT-MT attachment regulation, we agree that we do not have a similar way to inhibit PP2A-B56 activity to rescue hyperstable microtubule attachment when MELT numbers are high. For this, we require a way to rapidly inhibit PP2A-B56 activity after attachments have formed, something that is not technical feasible at the present. We can also not say for certain that reduced MELT numbers destabilize microtubule due to lack of PP2A, however we feel this is the most like interpretation for the following reasons. The phenotype of removing PP2A from BUBR1 or removing the MELT from KNL1 (along with all associated factors), is identical: mutant cells have comparable chromosome misalignment due to unattached kinetochores (compare 2F-I with 5A-D). Therefore, the additional factors lost by removing the MELTs cannot be having such a strong impact in KT-MT attachment. The obvious factor that could affect attachment strength is again BUB1, via Aurora B recruitment to centromeres. However, loss of BUB1 (after MELT removal) is predicted to enhance attachment stability (reduced Aurora B) and not decrease it, as we observe. So, whilst we cannot definitely conclude that modulating MELT number affect attachment stability via PP2A, we feel that this is certainly the most likely explanation. We will state this clearly in the revised text.

    Description of analyses that authors prefer not to carry out

    “SAC strength of BubR1 WT, ΔC and B56γ was analysed in the presence of nocodazole + MPS1i. It would be interesting to see what the phenotypes are without MPS1i [Reviewer 1]”

    In the absence of MPS1i basal MELT phosphorylation increases (DC) or decreases (B56g) as predicted (Figure 2d; compare timepoint 0 all conditions). This does not cause any change to SAC strength when all kinetochores are unattached in nocodazole (not shown). The sensitize SAC assay (nocodazole + MPSi) has been used by many groups (originally Santaguida et al, 2011; Saurin et al, 2011), because it reduces SAC signals from all unattached kinetochores which would otherwise produce a saturated response. In this case, we specifically chose a dose of MPS1 inhibitor that gave a partial SAC response from which we could observe either strengthening or weakening – a key point of the assay. Indeed, this showed that the SAC was strengthened (DC) or weakened (B56g), as predicted (Figure 2E). The only other way to do this, which has been used by some in the literature, is to use a low dose of nocodazole which prevents all kinetochore from signaling to the SAC. We specifically wanted to avoid this situation because then you cannot untangle the effects on SAC and KT-MT attachment stability – this was crucial in our case.

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    Referee #3

    Evidence, reproducibility and clarity

    A dividing cell relies on both error correction and spindle assembly checkpoints (SACs) to ensure accurate chromosome segregation. The PLK1-PP2A pair resides at the interface of the two pathways and makes these pathways robust and responsive. By generating fusion proteins and mutagenesis to perturb PLK1 and PP2A activities in HeLa cells, the authors found that both enzymes constitute a negative feedback loop on the scaffold BubR1. The docked PLK1 phosphorylates the PP2A binding site to recruit PP2A, while the PLK1 docking site is dephosphorylated after PP2A recruitment. They also manipulate kinetochore recruitment of PP2A to show effects on the SAC and on kinetochore-microtubule attachments. Independently, by varying active MELT repeats on KNL1, the authors found that decreasing MELT repeats weakens the SAC with unstable attachments, while increasing MELT repeats strengthens the SACs with hyperstable attachments. Thus, the recruited level of BubR1 determines SAC strength and attachment stability. The data included in this paper are solid, and the concepts are interesting, but these potential strengths of the manuscript are obscured because the logical progression and presentation are difficult to follow.

    Major comments:

    1. The first three paragraphs of the introduction lead up to the question: "Why the same phosphatase complex is used for both process is still not clearly understood". One conceptually simple answer is that the SAC should be silenced as attachments are stabilized, so it makes sense to use the same enzyme to accomplish both tasks. If the authors have something else in mind, they should clarify.
    2. The argument linking the negative feedback loop to biological functions is not straightforward. The authors provide evidence in Figure 1 for regulatory pathways between docked PLK1 and bound PP2A. However, their assays in Figure 2 bypass the feedback loop by directly modulating PP2A activity. These experiment supports an argument that the kinase/phosphatase activity balance is important, but do not address the feedback loop specifically (which could potentially be done using mutations that disrupt the feedback regulation). The claim that "a homeostatic feedback loop maintains an optimal balance of PLK1 and PP2A on the BUB complex" is too strong because there is no direct evidence connecting the feedback loop to optimal function.
    3. The paragraph starting with "Given the roles of PLK1 and PP2A ...": How does the kinase-dominant situation destabilize KT-MT attachments? Is it by inhibiting PP2A or by phosphorylating some kinetochore components? The former pathway is unclear because the authors show that PLK1 promotes PP2A recruitment in Figure 1.
    4. The paragraph starting with "In summary, a balanced recruitment of PLK1 and PP2A ...": What is meant by "phosphorylation sites that block KT-MT attachment" (used twice in the paragraph)? Block means prevent binding, as opposed to activities that destabilize attachment by promoting unbinding. Which do the authors mean? The following is also unclear: "kinetochores are no longer responsive to MT attachment". If attachment is blocked, as stated in the previous sentence, then what does it mean to say that kinetochores are not responsive to attachment (which never occurred if it was blocked)?
    5. Figures 1-2 and Figures 3-5 are separate concepts, but this is never explained clearly in the manuscript. Specifically, Figures 1-2 focus on the antagonism between PLK1 and PP2A activities, whereas Figures 3-5 focus on changing both activities in the same direction (either increase or decrease). There is no transition from one part to the other. Both concepts should be explained and the hypotheses stated clearly.
    6. Several words are used ambiguously. "Homeostasis" is vague, and it is unclear what exactly the authors mean. As discussed above, the meaning seems different for figures 1-2 vs figures 3-5. "Reciprocal changes" is also unclear (and seems misleading) because the perturbations of PLK1 and PP2A levels are in the same direction (more MELT motifs means more binding sites for both). For "preserved" (description of figure 4F), it's unclear if the authors refer to the Bub1 and BubR1 levels at metaphase or the change between prometaphase and metaphase. The authors should clarify what they mean and how it is measured.
    7. "too much kinase-phosphatase module would cause a strong SAC and hyperstable KT-MT attachment, and too little would cause a weak SAC and hypostable KT-MT attachments": The reasoning behind these predictions is not clear. For example, why does high phosphatase not silence the SAC as explained earlier in the manuscript?
    8. The mitotic exit assay in Figure 4G is hard to interpret. Mitotic duration depends on establishing correct attachments and then silencing the SAC. 19xMELT could affect both. A better measurement of SAC silencing would be time from metaphase alignment to anaphase.
    9. The paragraph starting "In summary, PLK1 is able to phosphorylate MELTs to recruit Bub1": The authors should clarify what was already known and what advance they are making. Similarly, the sentence starting with "Therefore, the number of MELT motifs ..." should also clarify the advance relative to previous findings.

    Minor comments:

    1. Please include pages numbers (and line numbers are also helpful).
    2. Previous literature (PMID 17785528) suggests that phosphorylation of pT620 is important for KT-MT regulation but not SAC signaling. Can the authors comment on this?
    3. SAC silencing seems more appropriate than "mitotic exit" in the last sentence of the second paragraph.
    4. Figure 1 would be clearer with images and the relevant quantifications together in the same panel.
    5. In the first paragraph of Results, the authors primarily explain the impacts of PP2A on SAC silencing but not on KT-MT attachment, even though the topic sentence seems equally weighted to both.
    6. Some terms are not defined when first introduced, such as the KARD domain and B56gamma.
    7. Figure 1JK: how were mitotic cells enriched and harvested?
    8. Figure 2C: a log scale may help show changes in both directions.
    9. Chromosome alignment assays in Figure 2 are not so informative because perturbation either way can generate misaligned chromosomes. The primary figures are dense with data, so these results can be made supplemental.
    10. Figure 3F: can the authors comment on the B56gamma decrease between nocodazole and MG132 conditions?
    11. Figure 4A: the data are difficult to interpret as presented. It is not clear whether SAC signaling changes monotonically with number of MELT repeats. It would be better to plot MELT number vs a summary statistic (such as time to 50% mitotic exit or something else that the authors find informative).
    12. Figure 6. "uncouplr" should be "uncouple".
    13. Missing references in bibliography: Ghongane et al 2014, Roy et al. 2020.

    Significance

    The proposed model is conceptually significant, and this mechanistic work bridges several previous findings. Biochemical studies suggest that KNL1 harbors PLK1-PP2A and Bub complex, and functional studies suggest that truncation on MELT motifs generates mitotic errors (PMID 24363448, 24344183). Furthermore, biochemical and functional assays suggest that PLK1 is a key regulator of the SAC (PMID 33125045). In this work, the authors link functional consequences to biochemical interactions among KNL1, BubR1, PLK1, and PP2A, which is an advance.

    This work should appeal to the kinetochore and cell cycle communities, but the logical flow needs to be improved.

    My most relevant expertise is in mitotic kinases and regulation of kinetochore microtubules.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript by Corno et al the authors investigate how the kinase/phosphatase balance is regulated at kinetochores during mitosis. When kinetochores are unattached, mitotic kinases signal through the spindle assembly checkpoint (SAC) to prevent progression through mitosis. Once attachments have occurred, the activities of kinetochore phosphatases silence the SAC and stabilize kinetochore-microtubule attachments to promote exit from mitosis. Key regulators of this signaling are the BUB proteins Bub1 and BubR1, which recruit the kinase Plk1. BubR1 also recruits the phosphatase PP2A-B56 in a Plk1-dependent manner. By analyzing the recruitment of Plk1 and PP2A-B56 at unattached kinetochores, the authors uncovered a mechanism by which Plk1 and PP2A-B56 negatively regulate each other's recruitment, thereby maintaining an optimal kinase/phosphatase balance. Disrupting this balance can lead to either a hyperactive SAC or premature stabilization of erroneous attachments.

    Overall, this is a very careful study and the data is clear and consistent. My only comments are in regards to the interpretation of the Knl1 data on the manuscript.

    Comments

    • In figures 3 and 4, the authors engineer a system to manipulate the levels of BUBs at the kinetochore by modulating the number of MELT repeats on Knl1. For this, they mutate all 19 MELT repeats on Knl1 and then add back discreet number of MELT motifs that follow a consensus sequence. They find that a 6x mutant, which contains 6 repeats of this consensus MELT motif, is sufficient to rescue the functions of Knl1 on the SAC and on chromosome alignment. However, 12x and 19x MELT repeat versions show an increased SAC response and increased stability of kinetochore-microtubule attachments. The authors interpret this as a result of increased kinetochore levels of BUBs, and therefore, increased levels of both Plk1 and PP2A-B56 (Fig. 3). However, from their representative images in Fig. S3A, it does not appear as if BUB levels are significantly increased in cells expressing the 12x and 19x Knl1 mutants, compared to wild-type controls. Considering that in the 12x and 19x mutants Knl1 recruitment is reduced by more than half of controls (Fig. S3B) and because the authors normalized their BUB kinetochore intensity levels by the Knl1 values, it makes it seem as if BUB kinetochore levels are increased in the cell under these conditions. While in the 12x and 19x mutant conditions there are more molecules of BUBs per Knl1, the overall BUB levels are the same as in wild-type controls. Since the MELT repeat used throughout the paper is a consensus sequence that is likely optimal for BUB binding, it is possible that the phenotypes of the 12x and 19x mutants are explained because of an increase in the affinity of BUBs for Knl1 rather than overall levels. This would also help explain why Knl1 and BUBs are observed at the spindle midzone in the 19x mutant (Fig. S4). To distinguish between these possibilities, the authors might consider doing FRAP experiments using fluorescently labelled Bub1 or BubR1 and measure BUB protein dynamics at kinetochores.
    • Also in regards to the 12x and 19x mutants, because these reduce Knl1 kinetochore levels, this fact alone might explain some of the observed phenotypes, such as the mild defects in chromosome alignment (Fig. S5).

    Significance

    This is a significant advancement in our understanding of how the spindle assembly checkpoint is regulated by kinases and phosphatases at the kinetochore. The authors used very precise manipulations to dissect how these components are balanced and they uncovered a very interesting negative feedback mechanism. They also provided significant evidence for the importance of this balance in normal mitotic progression. This work will be of broad interest to cell biologists, as well as cancer biologists.

    My expertise is on the fields of cell biology, mitosis and cell division mechanisms.

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    Referee #1

    Evidence, reproducibility and clarity

    The proper segregation of chromosomes during mitosis depends on correct kinetochore-microtubule (KT-MT) attachments which is monitored by the spindle assembly checkpoint (SAC). The Bub complex composed of the Bub1-Bub3 and BubR1-Bub3 complexes plays an important role in regulating KT-MT by recruiting Plk1 and PP2A-B56 as well as the SAC components Mad1/Mad2 and Cdc20. The Bub complex is recruited to kinetochores by Bub3 binding directly to phosphorylated MELT repeats on KNL1. The MELT repeats are predominantly phosphorylated by Mps1 but can also be phosphorylated by Plk1.

    Here the authors investigate the Plk1 and PP2A-B56 module on BubR1 further. It is already known that Plk1 is recruited to BubR1 T620 (a Cdk1 site) and that this is important for proper chromosome segregation. Furthermore, BubR1 binds to PP2A-B56 through and LxxIxE motif that is phosphorylated by Plk1 to stimulate binding. The main message of the paper is that the Plk1-PP2A-B56 module on BubR1 is crucial for integrating SAC and KT-MT attachments and that a homeostatic negative feedback loop between Plk1 and PP2A-B56 exists to limit the levels of these enzymes. As outlined below I do not think that their experimental evidence/setup can be used to draw these conclusions. Overall, I have limited comments on the technical execution of experiments as this is overall done well but more on interpretations of results and what conclusions can be drawn. Also, most of the phenotypes/observations are consistent with the literature and not suprising.

    Major comments:

    The authors generate a scenario where high levels of Plk1 are recruited to BubR1 by removing the entire C-terminus to remove the PP2A-B56 binding site and a situation of high PP2A-B56 by replacing the BubR1 C-term with B56gamma. Firstly, these are crude mutants generating extreme situations - specifically the fusion of B56gamma which likely creates artificially high levels of BubR1-B56gamma making it difficult to make conclusions on the physiological levels. These mutants are then analysed in a number of assays and phenotypic consequences analysed in the presence of nocodazole + Mps1 to evaluate SAC strength. It would be interesting to see what the phenotypes are without Mps1 inhibition. I cannot see in any way how the authors from two end points (high kinase or high phosphatase) can reach a conclusion that a homeostatic negative feedback loop exists between these enzymes that is critical for integrating SAC and KT-MT interactions. They can only conclude on what happens if you have too much kinase or phosphatase but no data are addressing what happens if you manipulate the cross-talk on BubR1. The experiments to do must be to carefully tune the proposed cross talk and then monitor what happens. This can be done by bypassing Plk1 regulation of the PP2A-B56 binding site or increasing the strength of the Plk1 binding site. Furthermore, there is no data to show that cross-talk is changing in response to changes in KT-MT attachment status - is the levels and activities of Plk1/PP2A-B56 on BubR1 changing. Yes Plk1 and PP2A-B56 can regulate each other on BubR1 but is this regulated as proposed.

    My second comment relates to the fact that the two parts of the paper are not directly linked although the authors try to do this. They nicely manipulate the MELT repeats on KNL1 to change the number of Bub complexes. However, they cannot directly link the data to changes in Plk1 and PP2A-B56 levels only as many other things are changing. By increasing MELT numbers Bub complex and Mad1/Mad2 levels increase as well as an example and this makes interpretations complicated. To me these experiments are not addressing the main conclusions of the paper.

    Specific comments:

    1. I would caution the interpretation of phenotypes being suppressed by Plk1 inhibitors. This does not address whether it is BubR1 bound Plk1 that is specifically affected - several Plk1 substrates could be contributing. Similar for Mad1 phosphorylation they cannot conclude that it is Plk1 bound to BubR1 phosphorylating Mad1.
    2. On page 4 the authors write: "We sought to modulate MELT numbers in a way that would allow BUB complex levels to be increased or decreased in a graded manner, thereby causing reciprocal changes to PLK1 and PP2A levels." I do not see how this will result in reciprocal levels as total Bub complex levels are increased.
    3. Page 7 first paragraph authors write: "remained high despite inhibition of Mps1". This is not completely correct as levels are dropping dramatically after 5/10 min of Mps1 inhibition. Total drop seems more than WT situation.
    4. Page 7 second paragraph authors write: "implying the elevated PLK1 in this situation (Figure 3E) is also able to better amplify MELT signalling..." This cannot be concluded as the starting levels are so much higher in 19XMELT than WT and there is no data to show this is due to more Plk1 bound to BuBR1.
    5. Page 7 second section authors write: "Figure 5B shows that KNL1deltaMELT causes severe chromosome misalignments, as expected, given the PP2A-B56 binding to KNL1 is inhibited in this situation". Multiple things are changing so this cannot be interpreted only as a readout of PP2A-B56. With no MELTs there is no recruitment of SAC proteins.
    6. Page 7 second section authors write: "Therefore, the number of MELT motifs is crucial for determining the stability of KT-MT attachments, most probably by setting the levels of PP2A-B56..." Again many things are changing so impossible to interpret data in light of PP2A-B56.
    7. One possibility not mentioned by authors is that PP1 bound to KNL1 cannot act as efficiently on some MELT repeats and that the dephosphorylation by PP1 of the 19xMELT is different from KNL1 WT which would impact on their results.

    Significance

    See above.

  6. Version published to 10.1101/2022.05.22.492960v2 on bioRxiv
  7. Version published to 10.1101/2022.05.22.492960v1 on bioRxiv