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Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2008 Oct 24; Vol. 283 (43), pp. 28996-9003. Date of Electronic Publication: 2008 Aug 16. - Publication Year :
- 2008
-
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.
- Subjects :
- Cell Adhesion
Cell Line
Fluorescence Resonance Energy Transfer
Humans
Kinetics
Models, Biological
Models, Molecular
Models, Statistical
Mutagenesis
Phosphorylation
Protein Structure, Quaternary
Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
Static Electricity
Calcium-Binding Proteins chemistry
Mutation
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 283
- Issue :
- 43
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 18708665
- Full Text :
- https://doi.org/10.1074/jbc.M804782200