title: Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.
abstract: Activation of furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER-localized soluble furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed.
authors.affiliation: Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland 97201-3098, USA.
authors.forename: E D
authors.initials: ED
authors.lastname: Anderson
authors.forename: J K
authors.initials: JK
authors.lastname: VanSlyke
authors.forename: C D
authors.initials: CD
authors.lastname: Thulin
authors.forename: F
authors.initials: F
authors.lastname: Jean
authors.forename: G
authors.initials: G
authors.lastname: Thomas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D045683:Furin; D005982:Glutathione Transferase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016324:Sequence Tagged Sites; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000201:enzymology; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014616:Vaccinia virus
chemical_list: D017931:DNA Primers; D004792:Enzyme Precursors; D009842:Oligopeptides; D010455:Peptides; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D005982:Glutathione Transferase; D013381:Subtilisins; D045683:Furin