Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition
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Evaluation Summary:
Xing and colleagues present a cryoEM structure of the protein phosphatase 2A (PP2A)-B56 holoenzyme in complex with protein phosphatase methyltransferase-1 (PME-1). The structure reveals that PME-1 blocks the substrate binding site of PP2A by inserting an unstructured loop. This unexpected inhibitory mechanism is also coupled to a large conformation change in the PP2A-B56 holoenzyme and PME-1. Combined with biochemical and cellular assays, the authors suggest how PME-1 can regulate p53-mediated DNA damage responses via inhibiting PP2A. This manuscript will be of importance for structural biologists as well as colleagues in the p53 field.
(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 #2 agreed to share their name with the authors.)
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Abstract
Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme–PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.
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Evaluation Summary:
Xing and colleagues present a cryoEM structure of the protein phosphatase 2A (PP2A)-B56 holoenzyme in complex with protein phosphatase methyltransferase-1 (PME-1). The structure reveals that PME-1 blocks the substrate binding site of PP2A by inserting an unstructured loop. This unexpected inhibitory mechanism is also coupled to a large conformation change in the PP2A-B56 holoenzyme and PME-1. Combined with biochemical and cellular assays, the authors suggest how PME-1 can regulate p53-mediated DNA damage responses via inhibiting PP2A. This manuscript will be of importance for structural biologists as well as colleagues in the p53 field.
(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. …
Evaluation Summary:
Xing and colleagues present a cryoEM structure of the protein phosphatase 2A (PP2A)-B56 holoenzyme in complex with protein phosphatase methyltransferase-1 (PME-1). The structure reveals that PME-1 blocks the substrate binding site of PP2A by inserting an unstructured loop. This unexpected inhibitory mechanism is also coupled to a large conformation change in the PP2A-B56 holoenzyme and PME-1. Combined with biochemical and cellular assays, the authors suggest how PME-1 can regulate p53-mediated DNA damage responses via inhibiting PP2A. This manuscript will be of importance for structural biologists as well as colleagues in the p53 field.
(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 #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors used a combination of biochemical assays and cryoEM to investigate the role of PME-1 in regulating PP2A, which revealed that PME-1 uses its unstructured loops to associate with the B-domain of the PP2A holoenzyme to regulate the function of the C-domain. This is a high quality work. This reviewer finds the later work involving p53 to be a helpful step in explaining the role the PME-1:PP2A interaction can have on important phosphorylation pathways, but I consider this aspect of the work to be very preliminary, especially given its rather minor effects. That said, the authors do not make claims that extend beyond the scope of the evidence they provide and thus I find the connection and discussion of PME-1, PP2A and p53 to be suitable on the whole.
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Reviewer #2 (Public Review):
The manuscript by Li et al is well-written and contributes an elegant cryo-EM structure of the PP2A-B56 holoenzyme, providing key structural rationale for holoenzyme demethylation and the inhibition of PP2A holoenzyme activity. A strength of the manuscript is the complementation of the structural data with a comprehensive biochemical/functional characterization demonstrating a mechanism for an oncogenic function of PME-1 in the regulation (inhibition) of p53 phosphorylation via PP2A-B56 holoenzymes under basal and DDR conditions.
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Reviewer #3 (Public Review):
PME-1 catalyzes the removal of carboxyl methylation of the PP2A catalytic subunit and negatively regulates PP2A activity. Like the PP2A methyltransferase LCMT-1, PME-1 was previously thought to act only on the PP2A core enzyme. However, in this study, the authors show that PME-1 can interact and demethylate different families of PP2A holoenzymes in vitro. They also report the cryo-EM structure of the PP2A-B56 holoenzyme in complex with PME-1. Their structure reveals that the substrate-mimicking motif of PME-1 binds to the substrate-binding pocket of B56 subunit, which tethers PME-1 to PP2A, blocks substrate-binding to PP2A, and promotes PME-1 activation and demethylation of PP2A holoenzyme. Their further mutagenesis and functional analyses indicate that cellular PME-1 function in p53 signaling is mediated by …
Reviewer #3 (Public Review):
PME-1 catalyzes the removal of carboxyl methylation of the PP2A catalytic subunit and negatively regulates PP2A activity. Like the PP2A methyltransferase LCMT-1, PME-1 was previously thought to act only on the PP2A core enzyme. However, in this study, the authors show that PME-1 can interact and demethylate different families of PP2A holoenzymes in vitro. They also report the cryo-EM structure of the PP2A-B56 holoenzyme in complex with PME-1. Their structure reveals that the substrate-mimicking motif of PME-1 binds to the substrate-binding pocket of B56 subunit, which tethers PME-1 to PP2A, blocks substrate-binding to PP2A, and promotes PME-1 activation and demethylation of PP2A holoenzyme. Their further mutagenesis and functional analyses indicate that cellular PME-1 function in p53 signaling is mediated by PME-1 activity towards PP2A-B56 holoenzyme. In summary, this study has provided significant insights into our understanding of PP2A regulation by PME-1, demonstrating that PME-1 not only demethylates the PP2A core enzyme, but also the holoenzyme to control cellular PP2A homeostasis.
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Author Response
Reviewer 1
The authors used a combination of biochemical assays and cryoEM to investigate the role of PME-1 in regulating PP2A, which revealed that PME-1 uses its unstructured loops to associate with the B-domain of the PP2A holoenzyme to regulate the function of the C-domain. This is a high quality work. This reviewer finds the later work involving p53 to be a helpful step in explaining the role the PME-1:PP2A interaction can have on important phosphorylation pathways, but I consider this aspect of the work to be very preliminary, especially given its rather minor effects. That said, the authors do not make claims that extend beyond the scope of the evidence they provide and thus I find the connection and discussion of PME-1, PP2A and p53 to be suitable on the whole.
Response: We greatly appreciate the positive …
Author Response
Reviewer 1
The authors used a combination of biochemical assays and cryoEM to investigate the role of PME-1 in regulating PP2A, which revealed that PME-1 uses its unstructured loops to associate with the B-domain of the PP2A holoenzyme to regulate the function of the C-domain. This is a high quality work. This reviewer finds the later work involving p53 to be a helpful step in explaining the role the PME-1:PP2A interaction can have on important phosphorylation pathways, but I consider this aspect of the work to be very preliminary, especially given its rather minor effects. That said, the authors do not make claims that extend beyond the scope of the evidence they provide and thus I find the connection and discussion of PME-1, PP2A and p53 to be suitable on the whole.
Response: We greatly appreciate the positive comments and the recognition of our work.
Reviewer 2
The manuscript by Li et al is well-written and contributes an elegant cryo-EM structure of the PP2A-B56 holoenzyme, providing key structural rationale for holoenzyme demethylation and the inhibition of PP2A holoenzyme activity. A strength of the manuscript is the complementation of the structural data with a comprehensive biochemical/functional characterization demonstrating a mechanism for an oncogenic function of PME-1 in the regulation (inhibition) of p53 phosphorylation via PP2A-B56 holoenzymes under basal and DDR conditions.
Response: We greatly appreciate the recognition and positive comments of our work.
Reviewer 3
PME-1 catalyzes the removal of carboxyl methylation of the PP2A catalytic subunit and negatively regulates PP2A activity. Like the PP2A methyltransferase LCMT-1, PME-1 was previously thought to act only on the PP2A core enzyme. However, in this study, the authors show that PME-1 can interact and demethylate different families of PP2A holoenzymes in vitro. They also report the cryo-EM structure of the PP2A-B56 holoenzyme in complex with PME-1. Their structure reveals that the substrate-mimicking motif of PME-1 binds to the substratebinding pocket of B56 subunit, which tethers PME-1 to PP2A, blocks substrate-binding to PP2A, and promotes PME-1 activation and demethylation of PP2A holoenzyme. Their further mutagenesis and functional analyses indicate that cellular PME-1 function in p53 signaling is mediated by PME-1 activity towards PP2A-B56 holoenzyme. In summary, this study has provided significant insights into our understanding of PP2A regulation by PME-1, demonstrating that PME-1 not only demethylates the PP2A core enzyme, but also the holoenzyme to control cellular PP2A homeostasis.
Response: We greatly appreciate the recognition and the positive comments on our work.
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