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

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A at Ser¹⁶ and by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at Thr¹⁷ β-Adrenergic stimulation and 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. Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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