Phosphorylation of mSin1-CRIM domain regulates protein stability and substrate selectivity.

The mechanistic target of rapamycin (mTOR) signaling plays critical roles in controlling mammalian cell behavior and function in response to environmental changes, and thereby it is important for maintaining tissue homeostasis and organism health. Dysregulation of mTOR signaling leads to a variety of human diseases, such as cancer, obesity, type II diabetes, and neurodegeneration. The mTOR signaling functions through two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which differ in their composition, structures, and functions. Considerable knowledge has been obtained regarding mTORC1 in recent years, while, in comparison, much less is known about mTORC2. Rictor and mSin1 are key mTORC2-specific proteins and essential for mTORC2 functions. Rictor is a scaffold protein in mTORC2 as Raptor in mTORC1, while mSin1 is considered to function for recruiting the substrate to the mTORC2 and its expression appears to be Rictor-dependent. However, exact mechanisms remain elusive. To explore the relationship between Rictor and mSin1 and their functions in mTORC2, we have generated RICTOR-knockout HEK293T cells by using the CRISPR-Cas9 system and revealed that mSin1 protein expression was substantially diminished in these cells and so was the mTORC2 activity, whereas mSin1 mRNA expression did not show any changes in the absence of RICTOR. Further analyses demonstrated that Rictor/mTORC2 is crucial for mSin1 protein stability, which may be attributed, at least partly, to the mTORC2-associated phosphorylation of mSin1-CRIM domain. More interestingly, this phosphorylation may also affect substrate recognition of mTORC2. The detailed mechanistic study regarding the influence of phosphorylated mSin1 on mTORC2 activity has been carried out and will be presented at the meeting. Our results suggest that mTORC2 may have an autoregulatory function and affect its substrate selection. Our findings thus advance our understanding in both regulation and function of Rictor/mTORC2, and also provide a potential insight for new therapies development in a number of human diseases. Citation Format: Yueh-Ho Chiu, Wei Cui. Phosphorylation of mSin1-CRIM domain regulates protein stability and substrate selectivity [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A09.