Abstract 12383: Phosphomimetic Mutations Enhance FXYD1 (Phospholemman) Oligomerization and Reduce Its Interaction with the Na/K-ATPase in Live Cells
FXYD1 (phospholemman), a small sarcolemmal protein, is an important substrate for both protein kinase A (PKA) and protein kinase C (PKC) activity in cardiac muscle. To determine the mechanisms by which FXYD1 phosphorylation alters its functional regulation of NKA, we quantified fluorescence resonance energy transfer (FRET) between FXYD1 and its regulatory target NKA in live cells. Phosphorylation of FXYD1 was mimicked by mutations S63E (PKA site), S68E (PKA/PKC site) or S63E/S68E (both sites). The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the FXYD1-NKA regulatory complex. All phosphomimetic mutations of FXYD1 changed the quaternary structure of the regulatory complex, and reduced the apparent affinity of the FXYD1-NKA interaction. Notably, the effects of individual phosphomimetic mutations were not additive, suggesting that the S63 and S68 phosphorylation sites are redundant. In addition, FXYD1-FXYD1 FRET experiments indicated that the decreased binding affinity of the FXYD1-NKA complex was likely due to increased oligomerization of FXYD1 phosphomimetic mutants. Distance constraints obtained by FRET suggest that the FXYD1 oligomer adopts a ”pinwheel” quaternary conformation, a structure that is slightly altered by phosphomimetic mutations. Photon-counting histogram analysis of FXYD1 oligomerization resolved two species with 4-fold different molecular brightness, indicating the major FXYD1 oligomeric species is a tetramer. Phosphomimetic mutations increased the population of FXYD1 tetramers, which is in harmony with the present FRET results. In conclusion, the data suggest that FXYD1 phosphorylation increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of the tetramer and regulatory complex.
- © 2010 by American Heart Association, Inc.