Nemazanyy I. Gene cloning and characterization of CoA synthase a novel binding partner of ribosomal S6 kinase

The main aim of this research was the identification of novel binding partners of S6K1 with the use of yeast two hybrid system. For screening of mouse embryo cDNA library the full length S6K1 was used as a bait. 28 primary positives were subjected for maiting assay. One clone was further confirmed as true-positive and identified by DNA sequencing. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains and can catalyze last two steps of CoA biothinthesis. Enzymatic activities responsible for each step of CoA biosynthesis have been purified from various mammalian sources and characterized. However, these studies have not been extended into protein sequence analysis and cDNA cloning of enzymes involved in the pathway of CoA biogenesis. Biochemical assays using wild type recombinant protein, dePCoA, 4'phosphopantenate and P32 gamma ATP confirmed that the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. These results confirm that the novel protein is a mammalian CoA synthase (CoASy) - a bifunctional enzyme containing the last two components of CoA biosynthesis. The association between S6K1 CoASy was further confirmed between native and transiently overexpressed recombinant proteins in HEK293 and MCF7 mammalian cells using immunoprecipitation experiments. With use of transiently overexpresed deletion mutants of CoASy and S6K1 in HEK293 cell with subsequent immunoprecipitation of protein complex the region of interaction was mapped to the C-terminal parts of both CoA synthase and S6K1. Transient expression studies and confocal microscopy allowed us to demonstrate for the first time that full-length CoASy is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to thecytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis with proteinase K, indicated that CoA synthase is localized on the mitochondrial outer membrane. These data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria. In vitro studies indicated that the interaction between CoASy and S6K does not affect enzymatic activities of both proteins and that CoA synthase is not a substrate for S6 kinase. The effect of S6K pathway inhibitors (rapamycin and LY294002) on CoAsy was investigated in Hek293 cells transiently transfected with CoAsy. No significant changes in CoAsy enzymatic activities (4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase) were found. In addition, we detected no considerable changes in S6K1 activity towards phosphorylation of S6 ribosomal protein as substrate when CoASy was overexpressed in HEK293 cells. This study uncovers a potential link between mTor/S6K signalling pathway and energy metabolism through CoA and its thioester derivatives. We can speculate that membrane-associated CoAsy may form a binding platform for the formation of a multi-enzyme complex, which may include S6K, mTor and other signalling molecules and to bring S6K to mitochondria for phosphorilation of it mitochondrial substrates.