title: A factor with a zinc- and phorbol ester-binding domain is necessary for endosome fusion.
abstract: An inhibitory effect of several zinc chelators on endosome fusion reconstituted in an in vitro system has been recently reported (A. Aballay et al., 1995, Biochem. J. 312, 919-923). The factor that requires zinc for its activity is still unknown. Since the regulatory domain of protein kinase C (PKC) contains cysteine-rich motifs which coordinate zinc, we suspected that PKC or a PKC-like protein might be that factor. To test this hypothesis, we studied the effect of calphostin C, a specific inhibitor of PKC that interacts with the cysteine-rich motif, and PMA (phorbol 12-myristate 13-acetate), an activator of several PKC isoforms that bind to the same region, on endosome fusion. Calphostin C inhibited endosome fusion in a zinc-regulated manner, whereas PMA enhanced endosome fusion. Moreover, fusion was strongly stimulated when both PMA and zinc were added together to zinc-depleted fusion reactions. Inhibitors of the catalytic domain of PKC had no effect on the assay suggesting that the kinase activity is not required. In contrast, a glutathione S-transferase fusion protein containing a cysteine-rich region of the regulatory domain of PKCgamma inhibited endosome fusion in a PMA-dependent manner. Western blot analysis demonstrated the presence of proteins containing PKC-like cysteine-rich regions that are released from endosomal fractions by zinc chelators. These results indicate that the previously proposed zinc-dependent factor required for endosome fusion could be either a PKC isoform or a protein containing the phorbol ester-binding domain of PKC.
authors.affiliation: Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, 5500, Argentina.
authors.forename: A
authors.initials: A
authors.lastname: Aballay
authors.forename: N G
authors.initials: NG
authors.lastname: Arenas
authors.forename: A F
authors.initials: AF
authors.lastname: Quest
authors.forename: L S
authors.initials: LS
authors.lastname: Mayorga
mesh_terms: D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine; D011992:Endosomes / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D009281:Naphthalenes / Q000494:pharmacology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009281:Naphthalenes; D010446:Peptide Fragments; D010617:Phenanthridines; D011993:Recombinant Fusion Proteins; C016299:chelerythrine; C087857:protein kinase C gamma; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C; D015032:Zinc; D003545:Cysteine; D013755:Tetradecanoylphorbol Acetate