The copper-binding, membrane-anchored, cellular prion protein （PrP~） has two constitutive cleavage sites producing distinct N- and C-terminal fragments （N1/C1 and N2/C2）. Using RK13 cells expressing either human P...The copper-binding, membrane-anchored, cellular prion protein （PrP~） has two constitutive cleavage sites producing distinct N- and C-terminal fragments （N1/C1 and N2/C2）. Using RK13 cells expressing either human PrPc, mouse PrPc or mouse PrP^C carrying the 3F4 epitope, this study explored the influence of the PrP^C primary sequence on endoproteolytic cleavage and one putative PrPc function, MAP kinase signal transduction, in response to exogenous copper with or without a perturbed membrane environment. PrPc primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulat- ed kinase 1/2 （ERK1/2） phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrP^C showed increased N1/C1 cleavage in response to copper alone, accompanied by specific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrP^C-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrPc harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrPc pri- mary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence appears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of PrP^C-related signal transduction in response to exogenous stimuli.展开更多
文摘The copper-binding, membrane-anchored, cellular prion protein （PrP~） has two constitutive cleavage sites producing distinct N- and C-terminal fragments （N1/C1 and N2/C2）. Using RK13 cells expressing either human PrPc, mouse PrPc or mouse PrP^C carrying the 3F4 epitope, this study explored the influence of the PrP^C primary sequence on endoproteolytic cleavage and one putative PrPc function, MAP kinase signal transduction, in response to exogenous copper with or without a perturbed membrane environment. PrPc primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulat- ed kinase 1/2 （ERK1/2） phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrP^C showed increased N1/C1 cleavage in response to copper alone, accompanied by specific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrP^C-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrPc harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrPc pri- mary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence appears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of PrP^C-related signal transduction in response to exogenous stimuli.
