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J. Biol. Chem., Vol. 280, Issue 50, 41332-41341, December 16, 2005

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ATPase Activity of p97/Valosin-containing Protein Is Regulated by Oxidative Modification of the Evolutionally Conserved Cysteine 522 Residue in Walker A Motif^*

Masakatsu Noguchi1, Takahiro Takata1, Yoko Kimura, Atsushi Manno1, Katsuhiro Murakami1, Masaaki Koike1, Hiroshi Ohizumi, Seiji Hori, and Akira Kakizuka2

From the Laboratory of Functional Biology, Kyoto University Graduate School of Biostudies and Solution Oriented Research for Science and Technology (JST), Kyoto 606-8501, Japan and the Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan

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 (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.

Received for publication, September 2, 2005 , and in revised form, October 17, 2005.

^* This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Ministry of Labor and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the 21st Century Center of Excellence (COE) Program of the Ministry of Education, Culture, Sports, Science, and Technology to Graduate School of Biostudies and Institute for Virus Research, Kyoto University.

² To whom correspondence should be addressed. Tel.: 81-75-753-7675; Fax: 81-75-753-7676; E-mail: kakizuka{at}lif.kyoto-u.ac.jp.

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