14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban

Julia Menzel, Daniel Kownatzki-Danger, Sergiy Tokar, Alice Ballone, Kirsten Unthan-Fechner, Markus Kilisch, Christof Lenz, Henning Urlaub, Mattia Mori, Christian Ottmann, Michael J. Shattock, Stephan E. Lehnart, Blanche Schwappach

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Abstract

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 β-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. β-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to β-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism.

Original languageEnglish
JournalScience signaling
Volume13
Issue number647
DOIs
Publication statusPublished - 1 Sep 2020

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    Menzel, J., Kownatzki-Danger, D., Tokar, S., Ballone, A., Unthan-Fechner, K., Kilisch, M., Lenz, C., Urlaub, H., Mori, M., Ottmann, C., Shattock, M. J., Lehnart, S. E., & Schwappach, B. (2020). 14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban. Science signaling, 13(647). https://doi.org/10.1126/scisignal.aaz1436