title: Short-term TNFα shedding is independent of cytoplasmic phosphorylation or furin cleavage of ADAM17.
abstract: Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Schwarz
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Broder
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Ansgard
authors.initials: A
authors.lastname: Helmstetter
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schmidt
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Isabell
authors.initials: I
authors.lastname: Yan
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Miryam
authors.initials: M
authors.lastname: Müller
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schmidt-Arras
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Becker-Pauly
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Koch-Nolte
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Hans-Willi
authors.initials: HW
authors.lastname: Mittrücker
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Rabe
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany. Electronic address: rosejohn@biochem.uni-kiel.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rose-John
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University Kiel, 24098 Kiel, Germany.
authors.forename: Athena
authors.initials: A
authors.lastname: Chalaris
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D000072198:ADAM17 Protein; D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D045683:Furin / Q000378:metabolism*; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D059748:Proteolysis; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D014409:Tumor Necrosis Factor-alpha; D045683:Furin; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse
keywords: A Disintegrin And Metalloprotease; ADAM; ADAM17; ADAM17(ex/ex) cells; ADAM17-hypomorphic cells; CHX; Cell surface trafficking; Cycloheximide; EGF; Epidermal Growth Factor; Furin; GPI; ICD; Intracellular domain; LPA; MAPK; PMA; Phosphorylation; TNF receptor; TNF-α; TNFR; TNFα; Tumor Necrosis Factor-α; glycosylphosphatidylinositol; intracellular domain; lysophosphatidic acid; mEF; mitogen-activated protein kinase; murine embryonic fibroblast; phorbol 12-myristate 13-acetate