Close

PP1 and PP2A use opposite phospho-dependencies to control distinct processes at the kinetochore

Richard J Smith, Marilia H Cordeiro, Norman E Davey, Giulia Vallardi, Andrea Ciliberto, Fridolin Gross, Adrian T Saurin

Posted on: 17 July 2019 , updated on: 18 July 2019

Preprint posted on 5 May 2019

Article now published in Cell Reports at http://dx.doi.org/10.1016/j.celrep.2019.07.067

How do highly similar phosphatases achieve functional specificity? The motifs recruiting them reveal more than you’d expect!

Selected by Angika Basant

Categories: cell biology

Background:

There are about 400 Ser/Thr kinases in the human genome but only 28 known Ser/Thr phosphatases (1, 2). How does this small group of phosphatases accurately regulate the phospho-state of hundreds of substrates?

PP1 and PP2A-B56 belong to the same PPP family of phosphatases. They share a similar 280 residue catalytic domain, the activities of which are comparable (3). Yet one does not appear to fully compensate the loss of the other in vivo (4), suggesting that each performs many unique functions. Why and how is a given phosphatase better-suited for certain roles over another? What features make it distinct?

This study addresses these questions in the context of the kinetochore. During spindle assembly, the kinetochore needs to perform two critical functions. It must (A) make stable, accurate attachments with microtubules and (B) signal the cell to maintain mitotic state until (A) has been achieved. These two functions are controlled by the phospho-states of the kinetochore protein Ndc80 and MELT motifs of the Knl1 protein respectively. Aurora B kinase phosphorylation of Ndc80 destabilises incorrect kinetochore-microtubule interactions. MELT motif phosphorylation by Mps1 kinase signals the cell to remain in mitosis. These two key phospho-modifications are also regulated by kinetochore phosphatases.

Both PP1 and PP2A-B56 are recruited to the Knl1 subcomplex on the kinetochore. PP1 directly binds the N-terminus of Knl1 to a RVSF motif. PP2A-B56 binds a LSPIIE motif of a Knl1 interactor. Interestingly, both these motifs are phosphorylated. And rather curiously, this has opposite effects on binding of the respective phosphatase. PP1 binding is reduced by phosphorylation of RVSF (5, 6), while PP2A-B56 binding is enhanced by LSPIIE phosphorylation (7, 8, 9).

Why are two similar phosphatases recruited to the same scaffold? Do they perform different functions? How are their individual activities regulated? Do their dissimilar binding patterns play a role?

Modified from Smith et al., 2019. KT, kinetochore, MT, microtubule. SAC, spindle assembly checkpoint to maintain mitotic state until chromosomes are properly aligned.

 

Key findings:

The authors first establish that the PP1 and PP2A-B56 do not perform identical functions at the kinetochore. Deleting LSPIIE, but not RVSF, results in increased Ndc80 phosphorylation and chromosome alignment defects. This implies that in a wild-type kinetochore, only PP2A-B56 directly regulates phospho-Ndc80.

Removal of either LSPIIE or RVSF results in mitotic arrest and high phospho-MELT. However, both phosphatases do not directly dephosphorylate MELT. PP2A-B56 is known to promote PP1 association to the kinetochore by dephosphorylating Knl1 (10). When these Knl1 phosphosites are mutated, removal of the LSPIIE motif no longer causes mitotic arrest, while deleting RVSF still results in arrest and high phospho-MELT. Collectively, these results indicate that only PP1 directly regulates mitotic arrest/exit. While PP2A-B56 indirectly facilitates PP1 recruitment, it does not dephosphorylate MELT motifs on wild-type kinetochores (even though it is detectable there).

Two hypotheses were tested to explain these differences. First – the identity of the phosphatase could be critical to each function. However, deleting the motif recruiting one and artificially recruiting the other in its place had no measurable effects.

Second, the authors considered whether the exact binding position of the phosphatase matters to function by say, controlling substrate accessibility. But deleting the motif recruiting either phosphatase and recruiting the same phosphatase to a different site on Knl1 did not impact its function. Ndc80 dephosphorylation occurred normally when PP2A-B56 was repositioned and MELT dephosphorylation was not impacted by PP1 repositioning.

If the two enzymes can functionally substitute for each other and their positions within the Knl1 subcomplex is immaterial to signalling, how are PP1 and PP2A-B56 restricted to distinct functions at the kinetochore?

As mentioned previously, a key difference between the two enzymes is the mechanism by which they are recruited to their respective motifs. To explore this aspect, the authors focused on a critical unexplained observation that despite being present at the kinetochore, PP2A-B56 does not dephosphorylate MELT motifs in the absence of PP1. They take advantage of a motif LPTIHE which binds PP2A-B56 with an affinity similar to LSPIIE but independently of phosphorylation. Amazingly, in the absence of RVSF (and thereby PP1), if the motif recruiting PP2A-B56 is changed to LPTIHE, PP2A-B56 is now able to substitute for PP1 and induce cells to exit mitosis.

The authors next construct a mathematical model for this network. It assumes that kinase activities involved in the system are fixed and that both PP1 and PP2A-B56 dephosphorylate the following substrates with the same kinetics, namely MELT, Ndc80, RVSP and LSPIIE. The only difference is that PP2A-B56 is recruited by phosphorylation of the motif it binds to, and PP1 is not. Strikingly, such a model is able to recapitulate their experimental findings.

Finally, in a wider search for such motifs, the authors find that RVxF and LxxIxE are present in ~700 proteins and a large fraction of these contain phosphorylatable residues in key positions. Quite interestingly, structural examination of PP1 and B56 reveals how a negatively charged surface on the former and a basic groove on B56 would explain the opposite behaviour towards a negatively charged phosphate in the motifs they bind.

What I like about this preprint:

This paper tackles a complex but widely-applicable problem with a nice combination of clear experiments, mathematical modelling and a wider analysis of the implicated motifs in other contexts. The key finding of this paper that inverse phospho-dependencies in phosphatase binding can create unique functional differences between similar enzymes, is unexpected and novel.

Questions for the authors:

  1. How critical are binding kinetics to the manifestation of these feedback loops? For example, if FRAP recovery of PP2A-B56 accumulation at the kinetochore was compared between LSPIIE- and LPTIHE-driven binding, would they be significantly different? And does that matter for the striking result seen in Figure 4E?
  2. Among the validated proteins with RVxF/LxxIxE motifs that are phosphorylated, are the predicted binding partners more often kinases and phosphatases? That is, is such phospho-dependence more likely to impact a certain type of signalling network and do you predict that tyrosine kinases/phosphatases may be regulated this way, albeit with different motifs?

References: 

  1. Almo et al., Struct. Funct. Genomics, 2007
  2. Manning et al., Science, 2002
  3. Ingebritsen and Cohen, Eur J Biochem, 1983
  4. Kauko et al., bioRxiv, 2018
  5. Kim et al., J Bio Chem, 2003
  6. Nasa et al., Sci Signal, 2018
  7. Wang et al., Protein Cell, 2016
  8. Wang et al., Structure, 2016
  9. Hertz et al., Mol Cell, 2016
  10. Nijenhuis et al., Nat Cell Biol, 2014

Tags: kinetochore, phosphatases, signalling networks

doi: https://doi.org/10.1242/prelights.12195

Read preprint (3 votes)

Author's response

Adrian Saurin shared

Hi Angika

Thanks for this great description of our work and very insightful questions. This is an excellent forum to discuss the findings so I’ll begin by responding to your request to provide some further insights into the work and how it evolved. Then I’ll finish by answering your questions.

One of the things we liked the most about this study was that it was both surprising at first and obvious in the end. It was surprising because phosphatases achieve specificity without actually doing anything obviously “specific”. Instead, they manage to exert control over different processes because they are inversely dependent of phosphorylation, which means that removal of either one produces very different network effects. Therefore, in the end, the key difference turns out to be one thing that we have known about for some time – their opposite phospho-dependencies. While this may appear fairly obvious in hindsight, I can tell you on behalf of Richard and Marilia – who did the majority of experimental work for this paper – that this was certainly not obvious at first! In fact, we were all convinced that switching the phosphatases or their positions would reveal a key difference, but 2 years of “negative” data told us otherwise. In the end, this negative data turned out to be important because when we discovered that phospho-regulation was critical we were then able to conclude that this was the main (perhaps even only) difference at kinetochores. This is important because if kinetochores select different phosphatases based on this one key feature then perhaps other pathways do as well – the SLiM analysis performed by Norman adds weight to this hypothesis.

One final note, I would like to give special praise to Fridolin and Andrea who performed all the mathematical modelling for this study. We had numerous skype calls and the model evolved continuously alongside the experiments. In the end, it is used as a proof-of-principle to conceptualise how different network behaviours can arise from identical phosphatases that have opposite phospho-dependencies. What you don’t see in the paper though, is just how much the modelling helped shape our thoughts throughout this study. It was an incredibly valuable tool and hopefully it will continue to be that going forward. We hope that one day we’ll get towards a model that can predict kinetochore behaviour and no doubt that will be very different again from the one we present in this study.

In response to your questions.

1. The point you raise on binding kinetics is an important one because these are very likely to affect phosphatase function, and they may well be different for the stated examples (i.e. PP2A with vs without phosphoregulation). If this is the case however, then it would also be important to understand why these kinetics are different, because that may also be due to network-based effects. For example, dephosphorylation may drive phosphatase release, which is something we have discussed in a recent article (Gelens et al., Dev Cell 2018) and we are currently investigating further. If it does, then this creates even more complexity for the modelling.

2a. This is also a very good question, but not an easy one to answer at this stage since even if the motif is not present in an actual kinase/phosphatase it could localise the phosphatase to a complex containing these signalling enzymes. Some very good examples of this are KNL1 and RepoMan. In these cases multiple kinases and phosphatase converge on a signalling complex which implies that these complexes are major sites for signal integration.

2b. Regarding tyrosine kinases, then yes definitely. I think we’ll find that many other phosphatases use SLiMs that direct them to their site of action and allow different forms of regulation. It’s certainly an exciting time for phosphatase research considering there is still so much to be discovered!

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos

Estefania Sanchez-Vasquez, Marianne E. Bronner, Magdalena Zernicka-Goetz

Selected by 11 October 2024

Anchel De Jaime Soguero

Developmental Biology

Global coordination of protrusive forces in migrating immune cells

Patricia Reis-Rodrigues, Nikola Canigova, Mario J. Avellaneda, et al.

Selected by 10 October 2024

yohalie kalukula

Biophysics

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites

Joelle Lo, Katherine F. Vaeth, Gurprit Bhardwaj, et al.

Selected by 24 September 2024

Felipe Del Valle Batalla

Neuroscience

preLists in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close