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Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2011 Mar 18; Vol. 286 (11), pp. 9120-6. Date of Electronic Publication: 2011 Jan 10. - Publication Year :
- 2011
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Abstract
- Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.
- Subjects :
- Amino Acid Substitution
Cell Line
Humans
Membrane Proteins genetics
Mutation, Missense
Phosphoproteins genetics
Protein Binding
Protein Structure, Quaternary
Sodium-Potassium-Exchanging ATPase genetics
Membrane Proteins metabolism
Phosphoproteins metabolism
Protein Multimerization physiology
Sodium-Potassium-Exchanging ATPase metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 286
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 21220422
- Full Text :
- https://doi.org/10.1074/jbc.M110.198036