title: ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in Walker A motif.
abstract: Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.
authors.affiliation: Laboratory of Functional Biology, Kyoto University Graduate School of Biostudies and Solution Oriented Research for Science and Technology (JST), Kyoto 606-8501, Japan.
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Noguchi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Takata
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kimura
authors.forename: Atsushi
authors.initials: A
authors.lastname: Manno
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Murakami
authors.forename: Masaaki
authors.initials: M
authors.lastname: Koike
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ohizumi
authors.forename: Seiji
authors.initials: S
authors.lastname: Hori
authors.forename: Akira
authors.initials: A
authors.lastname: Kakizuka
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019143:Evolution, Molecular; D005434:Flow Cytometry; D017353:Gene Deletion; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D019636:Neurodegenerative Diseases / Q000378:metabolism; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry*; D010641:Phenotype; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry; D000074405:Valosin Containing Protein
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C505032:CFTR protein, human; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011994:Recombinant Proteins; C491497:UFD1 protein, human; D025801:Ubiquitin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D013912:Threonine; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol