Enzymatic and biochemical characterization of the Target Of Rapamycin (TOR) signaling pathway

Abstract

Target Of Rapamycin (TOR) is a master regulator of cell growth in eukaryotes. TOR is a large, highly conserved serine/threonine protein kinase that belongs to the family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR functions in two distinct conserved complexes: rapamycin sensitive TORC1 and rapamycin insensitive TORC2. Rapamycin interacts with its cofactor, FKBP12, to specifically inhibit TORC1 by unknown mechanisms. Inhibition of TORC1 results in a dramatic drop in anabolic processes such as ribosome biogenesis and an induction of catabolic processes such as macroautophagy. TORC1 signals to these different machineries in large part via its direct substrate, the AGC family kinase Sch9. To better understand its regulation and inhibition we have established in vitro conditions to assay TORC1 kinase activity using Sch9 as a substrate. In addition, we have studied the in vivo signaling events downstream of TORC1 by characterizing Rph1 protein as a novel TORC1 target. This has allowed us to characterize novel TORC1 substrates and inhibitors and raised new aspects and questions regarding TORC1 signaling.