Protein Kinase A (PKA) Phosphorylation of Na+/K+-ATPase Opens Intracellular C-terminal Water Pathway Leading to Third Na+-binding site in Molecular Dynamics Simulations

Hanne Poulsen,Poul Nissen,Ole G Mouritsen,Himanshu Khandelia

doi:10.1074/jbc.m112.340406

Hanne Poulsen, Poul Nissen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m112.340406

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Abstract

Phosphorylation is one of the major mechanisms for posttranscriptional modification of proteins. The addition of a compact, negatively charged moiety to a protein can significantly change its function and localization by affecting its structure and interaction network. We have used all-atom Molecular Dynamics simulations to investigate the structural consequences of phosphorylating the Na(+)/K(+)-ATPase (NKA) residue Ser(936), which is the best characterized phosphorylation site in NKA, targeted in vivo by protein kinase A (PKA). The Molecular Dynamics simulations suggest that Ser(936) phosphorylation opens a C-terminal hydrated pathway leading to Asp(926), a transmembrane residue proposed to form part of the third sodium ion-binding site. Simulations of a S936E mutant form, for which only subtle effects are observed when expressed in Xenopus oocytes and studied with electrophysiology, does not mimic the effects of Ser(936) phosphorylation. The results establish a structural association of Ser(936) with the C terminus of NKA and indicate that phosphorylation of Ser(936) can modulate pumping activity by changing the accessibility to the ion-binding site.

Highlights

There is an ongoing debate on whether and how the ion pump NKA can be regulated by protein kinase A (PKA)
The C-terminal mutants studied so far exhibit severely reduced sodium affinity and altered kinetics of extracellular sodium binding and release [4, 17, 42]. These effects are not observed for the S940E mutant, which has extracellular sodium affinity and relaxation rate constants similar to the wild-type values as determined from pre-steady-state currents after expression in Xenopus oocytes (Fig. 1). ␣2-Expressing oocytes were treated with forskolin and isobutylmethylxanthine to activate PKA as described previously [43, 44]
Molecular Dynamics (MD) simulations have been used to investigate the molecular effects of phosphorylation of Ser936 in NKA, a well documented PKA substrate

Summary

Background

Results: Phosphorylation of the PKA target Ser936 opens an intracellular ion pathway leading to the ion-binding sites Conclusion: PKA phosphorylation has a drastic impact on NKA structure and dynamics. We have used all-atom Molecular Dynamics simulations to investigate the structural consequences of phosphorylating the Na؉/K؉-ATPase (NKA) residue Ser936, which is the best characterized phosphorylation site in NKA, targeted in vivo by protein kinase A (PKA). The Molecular Dynamics simulations suggest that Ser936 phosphorylation opens a C-terminal hydrated pathway leading to Asp926, a transmembrane residue proposed to form part of the third sodium ion-binding site. Ser936 lies on the cytoplasmic loop connecting transmembrane (TM) and TM9, which is close to the C-terminal interaction network that controls a pathway to the ion-binding sites. We examine the effects of Ser936 phosphorylation on the C-terminal network, on the ion-binding sites, and on transmembrane structure and dynamics.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION