Studies into the mechanism of action of rapamycin, a potent anti- proliferative drug, have led to the discovery of a novel translational control pathway with critical roles in eukaryotic cell division. The central component of the pathway is the in vivo target of rapamycin, a protein that we call RAFT1 but is also known as FRAP or mTOR. RAFT1 is a large protein kinase related to the cell-cycle regulators ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs). The RAFT1 mechanism of action is poorly understood, but the anti- proliferative properties of rapamycin reveal an essential, drug- sensitive role in cell division for the RAFT1-mediated translation of specific mRNAs. To elucidate how RAFT1 regulates mRNA translation and cell cycle progression we propose to: (1) identify downstream components of the RAFT1 signaling pathway, (2) understand their in vivo function in RAFT1 signaling, and (3) identify the rapamycin- sensitive mRNAs whose translational inhibition leads to cell cycle arrest. We are taking two approaches to discover components of the pathway: a biochemical one to identify functionally important RAFT1 interacting proteins (RIPs) and a genetic one for suppressors of the anti-proliferative effects of rapamycin. We have already identified two components whose role in RAFT1 signaling we will analyze in detail: p60, a novel RIP that may regulate downstream stages of the pathway, and FRAT1, an oncogene whose overexpression, we have shown, confers resistance to the anti-proliferative effects of rapamycin. We have used a microarray-based strategy to identify mRNAs whose translation is inhibited by rapamycin in T-cells. With biochemical and genetic experiments we will determine how RAFT1 controls the translation of these mRNAs and address why their inhibition leads to cell cycle arrest. The anti-proliferative effects of rapamycin are of medical value and the drug is now in clinical trials for immunosuppressive and anti-cancer uses. Thus, our study of the RAFT1 signaling pathway will not only elucidate the workings of a critical regulator of cell division, but also explain how a clinically useful drug exerts its effects.
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