title: Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.
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.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois 60153, USA.
authors.forename: Qiujing
authors.initials: Q
authors.lastname: Song
authors.forename: Sandeep
authors.initials: S
authors.lastname: Pallikkuth
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D000254:Sodium-Potassium-Exchanging ATPase