Research ArticleBiochemistry

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

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Science Signaling  01 Sep 2020:
Vol. 13, Issue 647, eaaz1436
DOI: 10.1126/scisignal.aaz1436

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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.

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