Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.
To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRET(max)) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (K(D)) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRET(max) was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.
Hou
Zhanjia
Z
Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153, USA.
Kelly
Eileen M
EM
Robia
Seth L
SL
D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002448:Cell Adhesion; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D015233:Models, Statistical; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry; D055672:Static Electricity; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban