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

14-3-3 remembers the kinase: The membrane protein phospholamban decreases the duration of the cardiomyocyte Ca²⁺ signal and limits cardiomyocyte contraction by inhibiting the Ca²⁺ pump SERCA. Stimuli that cause the phosphorylation of phospholamban by PKA (which phosphorylates Ser¹⁶) or CaMKII (which phosphorylates Thr¹⁷) alleviate this inhibitory effect, resulting in increased force generation. Menzel et al. found that the 14-3-3 family of phosphoadaptor proteins protected phospholamban, specifically the pentameric form, from dephosphorylation. Although the PKA and CaMKII target sites are adjacent to each other, CaMKII-phosphorylated phospholamban interacted with 14-3-3 with higher affinity than did PKA-phosphorylated phospholamban. These results suggest that CaMKII would have a longer-lasting effect on SERCA activity than would PKA and show how different kinases acting on adjacent residues in a substrate can generate distinct biological outcomes. 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. [ABSTRACT FROM AUTHOR]