Autophagy gets a brake: DAP1, a novel mTOR substrate, is activated to suppress the autophagic process.

Autophagy, a highly regulated catabolic process, is controlled by the action of positive and negative regulators. While many of the positive mediators of autophagy have been identified, very little is known about negative regulators that might counterbalance the process. We recently identified deathassociated protein 1 (DAP1) as a suppressor of autophagy and as a novel direct substrate of mammalian target of rapamycin (mTOR). We found that DAP1 is functionally silent in cells growing under rich nutrient supplies through mTOR-dependent inhibitory phosphorylation on two sites, which were mapped to Ser3 and Ser51. During amino acid starvation, mTOR activity is turned off resulting in a rapid reduction in the phosphorylation of DAP1. This caused the conversion of the protein into a suppressor of autophagy, thus providing a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under conditions of nutrient deprivation. Based on these studies we propose the "gas and brake" concept in which mTOR, the main sensor that regulates autophagy in response to amino acid deprivation, also controls the activity of a specific balancing brake to prevent the overactivation of autophagy.

DAP1, a novel substrate of mTOR, negatively regulates autophagy.

Autophagy, a catabolic process responsible for the degradation of cytosolic components, is upregulated when nutrient supplies are limited. A critical step in autophagy induction comprises the inactivation of a key negative regulator of the process, the Ser/Thr kinase mammalian target of rapamycin (mTOR). Thus far, only a few substrates of mTOR that control autophagy have been identified, including ULK1 and Atg13, both of which function as positive mediators. Here we identify death-associated protein 1 (DAP1) as a novel substrate of mTOR that negatively regulates autophagy. The link of DAP1 to autophagy was first apparent in that its knockdown enhanced autophagic flux and in that it displayed a rapid decline in its phosphorylation in response to amino acid starvation. Mapping of the phosphorylation sites and analysis of phosphorylation mutants indicated that DAP1 is functionally silenced in growing cells through mTOR-dependent phosphorylations on Ser3 and Ser51. Inactivation of mTOR during starvation caused a rapid reduction in these phosphorylation sites and converted the protein into an active suppressor of autophagy. These results are consistent with a "Gas and Brake" model in which mTOR inhibition also controls a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under nutrient deprivation.

DAP5 promotes cap-independent translation of Bcl-2 and CDK1 to facilitate cell survival during mitosis.

DAP5 is an eIF4G protein previously implicated in mediating cap-independent translation in response to cellular stresses. Here we report that DAP5 is crucial for continuous cell survival in nonstressed cells. The knockdown of endogenous DAP5 induced M phase-specific caspase-dependent apoptosis. Bcl-2 and CDK1 were identified by two independent screens as DAP5 translation targets. Notably, the activity of the Bcl-2 IRES was reduced in DAP5 knockdown cells and a selective shift of Bcl-2 mRNA toward light polysomal fractions was detected. Furthermore, a functional IRES was identified in the 5'UTR of CDK1. At the cellular level, attenuated translation of CDK1 by DAP5 knockdown decreased the phosphorylation of its M phase substrates. Ectopic expression of Bcl-2 or CDK1 proteins partially reduced the extent of caspase activation caused by DAP5 knockdown. Thus, DAP5 is necessary for maintaining cell survival during mitosis by promoting cap-independent translation of at least two prosurvival proteins.