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J. Biol. Chem., Vol. 279, Issue 51, 52970-52977, December 17, 2004

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Ubiquitylation of Neuronal Nitric-oxide Synthase by CHIP, a Chaperone-dependent E3 Ligase^*

Hwei-Ming Peng, Yoshihiro Morishima, Gary J. Jenkins, Anwar Y. Dunbar, Miranda Lau, Cam Patterson, William B. Pratt, and Yoichi Osawa¶

From the Department of Pharmacology, the University of Michigan Medical School, Ann Arbor, Michigan 48109 and the Departments of Pharmacology and Cell and Developmental Biology, Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina 27599

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 µM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.

Received for publication, June 21, 2004 , and in revised form, September 17, 2004.

^* This work was supported by National Institutes of Health Grants ES08365 (to Y. O.), HL65619 (to C. P.), GM61728 (to C. P.), and DK31573 (to W. B. P.). 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.

^¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pharmacology, University of Michigan Medical School, Medical Science Research Bldg. III, Ann Arbor, MI 48109-0632. Tel.: 734-763-5797; Fax: 734-763-4450; E-mail: osawa{at}umich.edu.

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