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J. Biol. Chem., Vol. 280, Issue 28, 26383-26396, July 15, 2005

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Mitochondrial Dok-4 Recruits Src Kinase and Regulates NF-B Activation in Endothelial Cells^*

Seigo Itoh, Serge Lemay¶, Masaki Osawa, Wenyi Che, Yuntao Duan||, Andrew Tompkins||, Paul S. Brookes||, Shey-Shing Sheu||, and Jun-ichi Abe**

From the Center for Cardiovascular Research, University of Rochester, Rochester, New York 14642, the Department of Medicine, Division of Nephrology, McGill University Health Center, Montreal, Quebec H3A 2B4, Canada, and the ^||Department of Anesthesiology, Pharmacology, and Physiology and Mitochondrial Research Interest Group, University of Rochester, Rochester, New York 14642

The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH₂-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH₂-terminal deletion mutant of Dok-4 (Dok-4-(N11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4-(N1-99), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4-mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor- (TNF-)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4-Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF--mediated NF-B activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-B activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF--mediated ROS production and NF-B activation.

Received for publication, September 7, 2004 , and in revised form, April 19, 2005.

^* This work was supported by American Heart Association Postdoctoral Fellowship 0325769T (to S. I.), National Institutes of Health Grants HL-66919 and HL-65262, American Heart Association Grant-in-aid 0455783T (to J.-i. A.), National Institutes of Health Grants HL-33333, NS-37710, and C017688, New York State Department of Health Spinal Cord Injury Research Fund (to S.-S. S.), Canadian Institutes of Health Research Grant MOP-53308, and The Kidney Foundation of Canada (to S. L.). 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.

^¶ To whom correspondence may be addressed: Dept. of Medicine, Division of Nephrology, McGill University Health Center, Lyman-Duff Bldg., Rm. 228, 3775 University St., Montreal, Quebec H3A 2B4, Canada. Tel.: 514-398-2762; Fax: 514-843-2815; E-mail: serge.lemay{at}mcgill.ca.

^** To whom correspondence may be addressed: Center for Cardiovascular Research, Box 679, 601 Elmwood Ave., University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. Tel.: 585-273-1686; Fax: 585-273-1497; E-mail: jun-ichi_abe{at}urmc.rochester.edu.

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