title: Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.
abstract: 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.
authors: Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153, USA.
authors: Zhanjia
authors: Z
authors: Hou
authors: Eileen M
authors: EM
authors: Kelly
authors: Seth L
authors: SL
authors: Robia
mesh_terms: 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
chemical_list: D002135:Calcium-Binding Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban