Molecular noise filtering in the β-adrenergic signaling network by phospholamban pentamers

Koch, Daniel; Alexandrovich, Alexander; Funk, Florian; Kho, Ay Lin; Schmitt, Joachim P.; Gautel, Mathias

doi:10.1016/j.celrep.2021.109448

Cited by 6 publications

(9 citation statements)

References 95 publications

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“…Perhaps such a switch-like transition is one of the main functions of cMyBP-C multisite phosphorylation/dephosphorylation. Similar switch-like responses stemming from multisite (de-)phosphorylation have been described by us for phospholamban (47). In the case of cMyBP-C, however, not all phosphorylation site combinations might correspond to discrete functional states.…”

Section: Discussionsupporting

confidence: 80%

“…Substrate dephosphorylation by PP1, for example, is subject to regulation by feedforward loops involving e.g. PKA, PKC and inhibitor-1 (47, 56), whereas sarcomeric PP2A activity is regulated by B56-subunits e.g. via changed localization after β-adrenergic stimulation (20, 33, 57) and via Pak1 (58), which itself lies downstream of multiple receptor tyrosine kinases.…”

Section: Discussionmentioning

confidence: 99%

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The cardiac myosin binding protein-C phosphorylation state as a function of multiple protein kinase and phosphatase activities

Kampourakis

Ponnam

Koch³

2023

Preprint

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Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a crucial determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C's multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2A (PP1 and PP2A) and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and protein kinases A (PKA), C and RSK2. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by PKA. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results emphasize the importance of phosphatases for cMyBP-C regulation and prompt us to propose reciprocal relationships between cMyBP-C m-motif conformation, phosphorylation state and myofilament function.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

The cardiac myosin binding protein-C phosphorylation state as a function of multiple protein kinase and phosphatase activities

Kampourakis

Ponnam

Koch³

2023

Preprint

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“…Specifically, we showed that a fraction of PLB unbinds from SERCA in response to cellular Ca 2+ elevations during cardiac systole (contraction), and this dynamic fraction of PLB is rapidly incorporated into PLB pentamers. This process reverses itself during diastole (cardiac relaxation) as Ca 2+ levels fall, but we (18,19) and others (28) showed that the rate of PLB pentamer dissociation is slow. This limits the rate of return of PLB monomers to SERCA during diastole.…”

Section: Introductionmentioning

confidence: 65%

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling

Cleary

Teng

Kongmeneck

et al. 2023

Preprint

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The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB (87.2 ± 3.2% pentamers) when compared to WT-PLB (79.5 ± 0.8% pentamers). In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation traps PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca2+ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.

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“…To understand this unexpectedly slow rate for PLB–SERCA rebinding, we considered whether the rate of PLB–SERCA recovery could be limited by slow dissociation of PLB monomers from the PLB pentamer ( 23 , 24 ). Indeed, PLB–PLB FRET relaxation (unbinding of monomers from the pentamer) displayed a slow time course (τ = 5.2 ± 0.8 s) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The rates at which PLB exchanges between SERCA- and pentamer-bound pools must govern how quickly these complexes can redistribute in vivo . We ( 23 ) and others ( 24 ) have provided evidence that the exchange of PLB monomers from pentamers occurs slowly relative to rapid exchange from the SERCA regulatory complex, but the underlying kinetics of these binding events have not been definitively measured. Therefore, the degree to which these regulatory complexes may dynamically redistribute in the cardiac SR remains unclear.…”

mentioning

confidence: 99%

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump

Cleary¹,

Fang²,

Cho³

et al. 2022

Journal of Biological Chemistry

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Molecular noise filtering in the β-adrenergic signaling network by phospholamban pentamers

Cited by 6 publications

References 95 publications

The cardiac myosin binding protein-C phosphorylation state as a function of multiple protein kinase and phosphatase activities

The cardiac myosin binding protein-C phosphorylation state as a function of multiple protein kinase and phosphatase activities

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump

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