Analysis of the mTOR Interactome using SILAC technology revealed NICE-4 as a novel regulator of mTORC1 activity.

The evolutionarily conserved mechanistic target of rapamycin (mTOR) forms two functionally distinct complexes, -the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)-which differ in their subunit composition. Although the function of mTORC1 has been studied extensively, the interaction between mTORC1 and the ubiquitin-proteasome system (UPS) remains unclear. To facilitate a thorough understanding of the mechanismby which UPS regulates mTORC1 activity, steady isotope labeling with amino acids in cell culture (SILAC) technology was used to screen for potential mTORC1-interacting UPS members. Fourteen previously unknown proteins bound to mTOR in HEK293 cells with a SILAC ratio (heavy/light, H/L) above 2, five of which are components of the UPS. Subsequent immunoprecipitation analysis confirmed that ubiquitin-relevant protein 2-like (UBAP2L, also known as NICE-4) binds to both mTOR and Raptor, but not Rictor, suggesting that NICE-4 specifically interacts with mTORC1, but not mTORC2. Interestingly, NICE-4 is essential for basic mTORC1 activity in both HeLa cancer cells and HEK293 cells. In addition, NICE-4 depletion markedly suppressed proliferation of both HeLa and HEK293 cells as well as survival of HeLa cells. Collectively, these results revealed the identity of novel mTOR-interacting UPS proteins and established NICE-4 as a critical UPS member that maintains mTORC1 activity.