Phorbol ester-regulated cleavage of normal prion protein in HEK293 human cells and murine neurons.
Cellular prion protein (PrP(c)) undergoes a proteolytic attack at the 110/111 downward arrow112 peptide bond, whereas the PrP isoform (PrP(res)) that accumulates in the brain tissue in Creutzfeldt-Jakob disease reveals an alternate cleavage site at about residue 90. Interestingly, the normal processing of PrP occurs inside the 106-126 amino acid region thought to be responsible for the neurotoxicity of the pathogenic prions, whereas PrP(res) cleavage preserves this potentially toxic domain. Therefore, any molecular mechanisms leading to enhanced cleavage at the 110/111 downward arrow112 peptide bond could be of potential interest. We set up TSM1 neurons and HEK293 stable transfectants overexpressing the wild-type or 3F4-tagged murine PrP(c), respectively. Both mock-transfected and PrP(c)-expressing cell lines produced an 11-12-kDa PrP fragment (referred to as N1), the immunological characterization of which strongly suggests that it corresponds to the N-terminal PrP(c) fragment derived from normal processing. We have established that the recovery of secreted N1 is increased by the protein kinase C agonists PDBu and PMA in a time- and dose-dependent manner in both cell lines. In contrast, secretion of N1 remains unaffected by the inactive PDBu analog alphaPDD and by the protein kinase A effectors dibutyryl cAMP and forskolin. Overall, our data indicate that the normal processing of PrP(c) is up-regulated by protein kinase C but not protein kinase A in human cells and murine neurons.
Vincent
B
B
Institut de Pharmacologie Moléculaire et Cellulaire du CNRS, UPR411, 660 Route des Lucioles, 06560, Valbonne, France.
Paitel
E
E
Frobert
Y
Y
Lehmann
S
S
Grassi
J
J
Checler
F
F
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003994:Bucladesine / Q000378:metabolism; D002273:Carcinogens; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000378:metabolism*; D011233:Precipitin Tests; D011328:Prions / Q000737:chemistry* / Q000378:metabolism*; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
D002273:Carcinogens; D010703:Phorbol Esters; D011328:Prions; D020033:Protein Isoforms; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000432:Methanol