Membrane-mediated dimerization potentiates PIP5K lipid kinase activity

  1. Scott D Hansen
  2. Albert A Lee
  3. Benjamin R Duewell
  4. Jay T Groves

  • Curated by eLife

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    Evaluation Summary:

    This paper is of interest for a wide range of readers who study the biology of lipid modifying enzymes, especially as it relates to interfacial reaction kinetics in biological membranes. This study aimed to obtain detailed biochemical insights into the mutual relationship between PI(4,5)P2 lipids and their kinase PIP5K, which engage in an exciting pattern-forming reaction on membranes. The authors find cooperative recruitment of PIP5K to the membrane, oligomerization-enhanced catalytic efficiency and indications of allosteric regulation. Although of very high interest and featuring mostly convincing data, there are concerns about the interpretation of whether the observed phenomenon is dimer specific or related to higher-order oligomerization. In addition, there are inconsistencies in the data presentation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P 2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P 2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer–dimer equilibrium. PIP5K monomers can associate with PI(4,5)P 2 -containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P 2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P 2 and membrane-bound kinase.

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  1. Version published to 10.7554/elife.73747 on eLife
  2. eLife

    Evaluation Summary:

    This paper is of interest for a wide range of readers who study the biology of lipid modifying enzymes, especially as it relates to interfacial reaction kinetics in biological membranes. This study aimed to obtain detailed biochemical insights into the mutual relationship between PI(4,5)P2 lipids and their kinase PIP5K, which engage in an exciting pattern-forming reaction on membranes. The authors find cooperative recruitment of PIP5K to the membrane, oligomerization-enhanced catalytic efficiency and indications of allosteric regulation. Although of very high interest and featuring mostly convincing data, there are concerns about the interpretation of whether the observed phenomenon is dimer specific or related to higher-order oligomerization. In addition, there are inconsistencies in the data presentation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    This paper presents a detailed molecular investigation into the behavior of PIP5K kinase, a membrane associated enzyme that catalyzes the production of PIP2 from PIP in cell membranes. Building on previous work on this system, the researchers use single-molecule fluorescence microscopy to study how the oligomeric state of PIP5K impacts membrane binding, PIP phosphorylation, and compositional patterning of lipid domains leading to stochastic bistability in membranes.

    A highlight of this study is the extensive experimental approaches that combine various single-molecule analyses, including diffusion and residence time distributions, as well as macroscopic measurements of membrane binding isotherms and PIP2 production. With this, it becomes evident that PIP5K exists in both monomeric and higher-order oligomeric forms, with the latter potentiating catalytic activity. This coupled to cooperative binding to the membrane linked to PIP2 production leads to a positive feedback system where patterning of the lipid composition emerges with stochastic bistable behavior, with oligomerization of the kinase acting as a modulating factor. This aspect of the research is interesting as it connects the higher-order oligomerization of the protein kinase to a means of modulating self-organization of the lipids within the cell membrane, a phenomenon that may be important for optimizing cellular signalling in biology.

    The majority of the studies are carried out carefully and with exquisite single-molecule approaches. However, a weakness of the study is that the ultimate conclusion of the activity linked specifically to dimerization is not clearly supported by the data. The results presented reflect a comparison of monomers vs. oligomers, without a clear identification of conditions where dimers persist. The mutation constructed to disrupt dimerization shifts the system to monomers with an associated decrease in catalytic activity. However, this finding does not provide a strong connection to the dimer state, but rather the loss of the effect when oligomerization is disrupted. Other properties of the protein may be impacted, such as stability and fold, as well as the overall binding propensity to the membrane. The catalytic activity measured per PIP5K molecule does indicate that an increased density for the wild-type protein leads to an increase in the rate of PIP2 production, providing evidence that oligomerization increases function. Yet, many of the results throughout the paper provide support for general oligomerization rather than dimerization, and so further investigation is needed in order to clarify the interpretation in what is otherwise an interesting system and study.

  4. eLife

    Reviewer #2 (Public Review):

    Hansen et al. investigates the catalytic behavior of phosphatidylinositol phosphate kinases (PIPKs), a family of enzymes that generate the regulatory lipid, phosphatidylinositol 4,5-bisphosphate (PIP2) of eukaryotic cells. In their previous studies the Authors showed the positive feed-back regulation of these enzymes by their reaction product, PIP2 using a clever methodology, namely the real-life fluorescent monitoring of the enzymatic activity in supported lipid bilayers. This time the Authors noted a substantial difference between the strength of dimerization of the type II (PIP5P 4-kinases) and the type I (PI4P 5-kinases) enzymes, the latter exhibiting very weak dimerization in solution in contrast to the stable dimer formation of the former. Using supported membrane bilayers, the Authors showed that at low protein density the type I enzyme (they used PIP5KB) followed the behavior described previously, namely membrane interaction determined by the presence of PIP2 in the bilayer and this behavior was the same for a mutant protein, unable to dimerize. However, at increased protein concentration, the PIP5KB enzyme started to form dimers, which increased its time of membrane residence, still dependent on PIP2. Furthermore, the Authors showed that dimerization had a major impact on catalytic activity, multiplying the positive feed-back effect described for the monomeric form. Lastly, they demonstrated the impact of the enhanced feed-back regulation under competitive reaction conditions (in the simultaneous presence of a PIP2 5-phosphatase) showing that the previously described bistable reaction product pattern is highly dependent on dimerization, which also increases the stochastic nature of product bistability in a competitive reaction setting. The Authors discuss the potential impact of these findings on the regulation of the enzyme in the real cellular setting.

    Strengths:

    This is an important study revealing a new layer of complexity in the interfacial kinetic behavior of an enzyme family that is central to the regulation of multiple cellular functions. The simplified in vitro set up allowed the Authors to examine in very exact terms the impact of protein dimerization on reaction kinetics and complex behavior under competitive reaction conditions. The in vitro methods are creative, the experiments are well done with appropriate controls and together with the data analysis convincingly support the Authors' conclusions.

    Weaknesses:

    1. The Discussion misses opportunities to relate the present findings to specific published observations: For example, it has been reported that type II PIP4KC knockout cells display increased PIP5K activity, presumably because of the heterodimerization of the proteins with the PIP5Ks, thereby reducing their activity (PMID: 31091439). An additional recent study described the regulation of the PIP5K by phosphatidylserine and cholesterol-rich domains (PMID: 31402097). Both of these studies raise questions that can be easily addressed by the reagents and methods described in the present study. Even if these studies are saved for the future, discussion of those published studies would emphasize the importance of the current findings in the context of the questions raised by them.

    2. As written, the paper is not always easily accessible to readers who are not experts in biophysical methodology and terminology. Some explanation may help general readers to follow the manuscript.

  5. eLife

    Reviewer #3 (Public Review):

    In their study "Membrane-mediated dimerization potentiates PIP5K lipid kinase activity", Hansen et al. aim to deepen their biochemical understanding of a fascinating self-organizing system the authors have previously been reporting on (Hansen et al., PNAS 2019), in particular, the regulation of PI(4,5)P2 lipids by the kinase PIP5K, which is itself recruited to the membrane by the PI(4,5)P2. From reconstitution studies on supported membranes investigated by TIRF microscopy, following elegant assays that have they previously developed, they conclude that PIPK5 activity is regulated by cooperative binding to and membrane-mediated dimerization of the kinase domain. Dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation and amplifies stochastic variation in the kinase reaction velocity, leading to stochastic geometry sensing that has been reported earlier.

    Overall, this is a beautiful biochemical system of great general interest. Also, the findings are plausible in the light of other pattern forming systems. However, the quality of both, the writing (with partly confusing annotations, inconsistencies, and missing clarity of what is actually reported on) and the data is extremely variable, giving the whole paper a somehow immature "patchwork" impression. Not the least, error bars are missing throughout the paper, and although both the protein/membrane system and the instrumental setup seem to be sufficiently well controlled, the quantitative aspect of this study could be greatly improved.

  6. eLife

    Author Response

    Reviewer 2

    Hansen et al. investigates the catalytic behavior of phosphatidylinositol phosphate kinases (PIPKs), a family of enzymes that generate the regulatory lipid, phosphatidylinositol 4,5bisphosphate (PIP2) of eukaryotic cells. In their previous studies the Authors showed the positive feed-back regulation of these enzymes by their reaction product, PIP2 using a clever methodology, namely the real-life fluorescent monitoring of the enzymatic activity in supported lipid bilayers. This time the Authors noted a substantial difference between the strength of dimerization of the type II (PIP5P 4-kinases) and the type I (PI4P 5-kinases) enzymes, the latter exhibiting very weak dimerization in solution in contrast to the stable dimer formation of the former. Using supported membrane bilayers, the Authors showed that at low protein density the type I enzyme (they used PIP5KB) followed the behavior described previously, namely membrane interaction determined by the presence of PIP2 in the bilayer and this behavior was the same for a mutant protein, unable to dimerize. However, at increased protein concentration, the PIP5KB enzyme started to form dimers, which increased its time of membrane residence, still dependent on PIP2. Furthermore, the Authors showed that dimerization had a major impact on catalytic activity, multiplying the positive feed-back effect described for the monomeric form. Lastly, they demonstrated the impact of the enhanced feed-back regulation under competitive reaction conditions (in the simultaneous presence of a PIP2 5-phosphatase) showing that the previously described bistable reaction product pattern is highly dependent on dimerization, which also increases the stochastic nature of product bistability in a competitive reaction setting. The Authors discuss the potential impact of these findings on the regulation of the enzyme in the real cellular setting.

    Strengths:

    This is an important study revealing a new layer of complexity in the interfacial kinetic behavior of an enzyme family that is central to the regulation of multiple cellular functions. The simplified in vitro set up allowed the Authors to examine in very exact terms the impact of protein dimerization on reaction kinetics and complex behavior under competitive reaction conditions. The in vitro methods are creative, the experiments are well done with appropriate controls and together with the data analysis convincingly support the Authors' conclusions.

    Weaknesses:

    1. The Discussion misses opportunities to relate the present findings to specific published observations: For example, it has been reported that type II PIP4KC knockout cells display increased PIP5K activity, presumably because of the heterodimerization of the proteins with the PIP5Ks, thereby reducing their activity (PMID: 31091439). An additional recent study described the regulation of the PIP5K by phosphatidylserine and cholesterol-rich domains (PMID: 31402097). Both of these studies raise questions that can be easily addressed by the reagents and methods described in the present study. Even if these studies are saved for the future, discussion of those published studies would emphasize the importance of the current findings in the context of the questions raised by them.

    We thank the reviewer for mentioning these important articles. We extended our discussion to connect the finding of these articles to the PIP5K regulatory mechanism described in our manuscript. The following statements have been added to the discussion section:

    “Although new molecular mechanisms concerning PIP5K activation have been revealed through single molecule characterization of PIP5K in vitro, it remains challenging to interpret how dimerization, PI(4,5)P2 binding, and interactions with peripheral membrane proteins regulate membrane localization of PIP5K in vivo. Complicating our interpretation of cellular localization, PIP5K can also reportedly interact with phosphatidylserine and sterol lipids, which modulate lipid kinase activity (Nishimura et al. 2019).”

    “Left unregulated, the PIP5K positive feedback loop has the potential to generate excessively high concentrations of PI(4,5)P2 in cells, which would be detrimental to numerous signaling pathways that rely on cellular PIP lipid homeostasis. New evidence suggests that, in vivo and in vitro, PIP4K can attenuate PIP5K activity through the formation of a membrane bound heterokinase complex (Wang et al. 2019; Wills et al. 2022). Deciphering the molecular basis of PIP4KPIP5K complex formation using single molecule in vitro measurements will be critical for determining both the mechanism of kinase inhibition and for generating separation of functions mutants that perturb this regulatory mechanism.”

    1. As written, the paper is not always easily accessible to readers who are not experts in biophysical methodology and terminology. Some explanation may help general readers to follow the manuscript.

    In our revised manuscript, we include additional description in the results and discussion sections to more clearly explain how our experiments were executed and the rationale for our interpretations.

  11. Version published to 10.1101/2021.09.21.461304v1 on bioRxiv