期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 283, 期 43, 页码 28996-29003出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M804782200
关键词
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资金
- National Institutes of Health Mentored Research Scientist Development Award [EB006061]
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 (FRETmax) 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 FRETmax was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.
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