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Abstract: TH-PO350

Regulation of SGK1 Phosphorylation and Activity by mTORC2

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic

Authors

  • Takagi, Enzo, University of California San Francisco, San Francisco, California, United States
  • Chen, Junliang, University of California San Francisco, San Francisco, California, United States
  • Wang, Feng, University of California San Francisco, San Francisco, California, United States
  • Saha, Bidisha, University of California San Francisco, San Francisco, California, United States
  • Pearce, David, University of California San Francisco, San Francisco, California, United States
Background


mTORC2 is a multi-subunit kinase complex comprising core components mTOR kinase, Rictor, mSin1 and mLST8, which has received increasing attention during recent years. The principal direct targets of mTORC2 phosphorylation include Akt (known for glucose regulation) and SGK1 (known for regulation of renal tubule ion transporters, particularly ENaC). However, structural information regarding mTORC2 has been limited, particularly in the context of SGK1 regulation. To gain greater structural insight into mTORC2 in the context of phosphorylating SGK1 we used cryo-EM complemented by mTORC2 kinase assays.

Methods


Core mTORC2 subunits were expressed in Expi293F cells and purified and cryo-EM grids prepared. Cryo-EM was performed using Krios at SLAC for high energy electrons for density maps of human mTORC2. Structures were solved for apo-complex at overall 3.23 Å resolution, and for co-complex with SGK1 at 3.38. Structural data predicted functional importance of mSin1-Arg-83, which was investigated through kinase assays in mSin1-deficient HEK293T cells transfected with WT and Arg-83-Ala mutant mSin1.

Results


The overall shape of mTORC2 in the SGK1 co-complex was similar to the apo-complex. Interestingly, the conformation of the mSin1 N-terminal domain was markedly altered in the SGK1 co-complex compared with the apo. First, an extended domain of mSin1 upstream of Arg-83 became unobservable, likely due to increased flexibility. Second, the Arg-83 side chain showed a large rotation toward a negatively charged patch within Rictor, appears to form a salt bridge with Rictor Asp-1679. Kinase assays performed using mTORC2 expressed in mSin1-deficient HEK-293T cells further supported the importance of mSin1 Arg-83: an Ala mutant of that residue selectively disrupted SGK1 phosphorylation, but not that of Akt.

Conclusion


These findings provide new structural and functional insights into mTORC2 substrate-specific activities and suggest new testable predictions, particularly with respect to mTORC2 regulation of SGK1 and ENaC. Further, these findings provide a potential avenue toward highly selective mTOR modulators with potential clinical utility.

Funding

  • NIDDK Support