Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components

Abstract

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis

“…are thus consistent with a model in which TRAP1 regulates the previously reported ability of mitochondrial c-Src to stimulate oxidative phosphorylation (14,15). Reduced/absent TRAP1 expression also correlates with increased cell motility/invasiveness that is sensitive to c-Src inhibition and ROS buffering strategies.…”

“…Miyazaki et al first reported that c-Src is located within mitochondria and affects energy metabolism by phosphorylating complex IV (14). Later studies confirmed that c-Src is present in the inner mitochondrial membrane/matrix (15). Although the mechanism by which TRAP1 regulates mitochondrial c-Src is not yet clarified, the cytosolic c-Src-HSP90 interaction provides a model to address this question.…”

“…Based on these data and on previous reports that mitochondrial c-Src stimulates complex IV activity and enhances oxidative phosphorylation (14,15), we speculated that the effect of TRAP1 on mitochondrial respiration may depend on its regulation of mitochondrial c-Src. To investigate this possibility, we confirmed that c-Src overexpression significantly increased the OCR of isolated HeLa cell mitochondria, but simultaneous overexpression of TRAP1 partially reversed this effect (Fig.…”

“…Plasmid expressing WT c-Src was purchased from Upstate Biotechnology. Plasmids expressing mitochondrially targeted c-Src constructs were kind gifts of Y. Homma (Department of Biomolecular Science, Fukushima Medical University School of Medicine, Fukushima, Japan) (15). MAFs were transiently transfected with TRAP1-GFP or empty-GFP plasmid using an Amaxa Nucleofector (Lonza), according to the manufacturer's protocol.…”

“…Unexpectedly, we found TRAP1 to be tyrosine phosphorylated (Fig. 3A, Left), which caused us to examine c-Src, a kinase reported to be present in mitochondria (14,15,(30)(31)(32)(33). We probed TRAP1 immunoprecipitates for c-Src association and we identified c-Src as a TRAP1-interacting protein in HCT116 cells (Fig.…”