| FKBP12-rapamycin associated protein (FRAP, also known as mTOR or RAFT) is the founding member of the PIK-related kinase family and functions as a sensor of physiological signals that regulate cell growth. Signals integrated by FRAP include nutrients, cAMP levels and osmotic stress, and cellular processes affected by FRAP include transcription, translation and autophagy. FRAP modulates the translational machinery by regulating p70 S6 kinase (p70S6K) and 4EBP by a poorly understood mechanism. Here, we show that FRAP constitutively restrains a PP2A isoform that dephosphorylates p70S6K and 4EBP upon activation. Nutrient deprivation, hyperosmotic conditions and rapamycin treatment result in modulation of FRAP leading to activation of this PP2A isoform and subsequent dephosphorylation of p70S6K and 4EBP. The mechanisms underlying the integration of signals such as nutrient deprivation and hyperosmotic conditions by FRAP are largely unknown. Here we show that exposure of cells to hyperosmotic conditions (and to glucose-deficient growth medium) results in rapid and reversible dissipation of the mitochondrial proton gradient. These results suggest that the ability of FRAP to mediate osmotic stress-response (and glucose deprivation-response) is via an intermediate mitochondrial dysfunction. We also show that in addition to cytosolic FRAP, a large portion of FRAP associates with the mitochondrial outer membrane (MOM). The results support the existence of a stress-sensing module consisting of mitochondria and MOM-associated FRAP. This module allows the cell to integrate a variety of stress-signals that affect mitochondrial function and regulate a growth-checkpoint involving p70 S6 kinase. To explore the role of mitochondria in integration and modulation intracellular signaling, we have initiated a chemical genetic approach to identify novel small molecule modulators of mitochondria. We have developed a novel high throughput assay to screen small molecule-libraries and identified a novel disruptor of mitochondrial membrane potential. As an alternative approach to modulate mitochondrial activity, we demonstrate a novel inducible gene expression system designed to be controlled by methallyl-rapamycin (MA-rap), a biologically inactive variant of rapamycin. This inducible expression system relies on MA-rap mediated assembly of an artificially designed DNA-binding domain ZFHD and transactivation domain p65 for expression control. This system is ‘ultra-quiet’ at baseline conditions. |
