PrPC-related signal transduction is influenced by copper, membrane integrity and the alpha cleavage site.
The copper-binding, membrane-anchored, cellular prion protein (PrP(C)) has two constitutive cleavage sites producing distinct N- and C-terminal fragments (N1/C1 and N2/C2). Using RK13 cells expressing either human PrP(C), mouse PrP(C) 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 PrP(C) function, MAP kinase signal transduction, in response to exogenous copper with or without a perturbed membrane environment. PrP(C) primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulated 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 PrP(C) 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 PrP(C) primary 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.
Haigh
Cathryn L
CL
Department of Pathology, The University of Melbourne, Parkville, Melbourne, 3010, Australia.
Lewis
Victoria A
VA
Vella
Laura J
LJ
Masters
Colin L
CL
Hill
Andrew F
AF
Lawson
Victoria A
VA
Collins
Steven J
SJ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D003300:Copper / Q000493:pharmacokinetics*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005372:Filipin / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008560:Membrane Fluidity / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D018621:PrPC Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism
D000900:Anti-Bacterial Agents; D010446:Peptide Fragments; D018621:PrPC Proteins; D017382:Reactive Oxygen Species; D003300:Copper; D005372:Filipin; D048049:Extracellular Signal-Regulated MAP Kinases