Mitochondrial Dok-4 recruits Src kinase and regulates NF-kappaB activation in endothelial cells.
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-(deltaN11-29)), 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-(deltaN1-99), 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-alpha (TNF-alpha)-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-alpha-mediated NF-kappaB activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-kappaB 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-alpha-mediated ROS production and NF-kappaB activation.
Itoh
Seigo
S
Center for Cardiovascular Research, University of Rochester, Rochester, New York 14642, USA.
Lemay
Serge
S
Osawa
Masaki
M
Che
Wenyi
W
Duan
Yuntao
Y
Tompkins
Andrew
A
Brookes
Paul S
PS
Sheu
Shey-Shing
SS
Abe
Jun-Ichi
J
D000111:Acetylcysteine / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004996:Ethidium / Q000031:analogs & derivatives / Q000494:pharmacology; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D007249:Inflammation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D010101:Oxygen Consumption; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C486625:DOK4 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C067883:dihydroethidium; D049452:Green Fluorescent Proteins; D019061:src-Family Kinases; D004996:Ethidium; D000111:Acetylcysteine