Phosphorylation of mammalian mitochondrial EF-Tu by Fyn and c-Src kinases

Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tu_mt) have been identified as possible Fyn and c-Src kinase targets. However, the phosphorylation of specific residues in EF-Tu_mt by these kinases and their roles in the regulation of protein synthesis are yet to be explored. In this study, we report the association of EF-Tu_mt with cSrc kinase and mapping of phosphorylated Tyr (pTyr) residues by these kinases. We determined that a specific Tyr residue in EF-Tu_mt at position 266 (EF-Tu_mt-Y266), located in a highly conserved c-Src consensus motif is one of the major phosphorylation sites. The potential role of EF-Tu_mt-Y266 phosphorylation in regulation of mitochondrial translation investigated by site-directed mutagenesis. Its phosphomimetic to Glu residue (EF-Tu_mt-E266) inhibited ternary complex (EF-Tu_mt•GTP•aatRNA) formation and translation in vitro. Our findings along with data mining analysis of the c-Src knock out (KO) mice proteome suggest that the SFKs have possible roles for regulation of mitochondrial protein synthesis and oxidative energy metabolism in animals.