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Proximity labeling of endogenous RICTOR identifies mTOR complex 2 regulation by ADP ribosylation factor ARF1

Amelia K Luciano1, Ekaterina D Korobkina1, Scott P Lyons2

  • 1Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.

The Journal of Biological Chemistry
|August 16, 2022
PubMed

Insights

Mechanistic target of rapamycin complex 2 (mTORC2) controls cell growth and survival. This study identified novel mTORC2 interacting proteins, including ARF1, revealing new insights into mTORC2 regulation and function.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Mechanistic target of rapamycin complex 2 (mTORC2) is a crucial regulator of cell metabolism, proliferation, and survival.
  • mTORC2 activity is stimulated by growth factors and phosphorylates key AGC kinases like AKT, SGK, and PKC.
  • The proteins interacting with mTORC2 to modulate its activity and localization are not well understood.

Purpose of the Study:

  • To identify novel proteins interacting with endogenous mTORC2 in living cells.
  • To investigate the potential nuclear localization and function of mTORC2.
  • To elucidate the role of ADP ribosylation factor 1 (ARF1) in mTORC2 regulation.

Main Methods:

  • Utilized BioID2 proximity labeling of endogenous RICTOR, an essential mTORC2 subunit, in live cells.
  • Performed mass spectrometry to identify RICTOR-proximal proteins.
  • Conducted imaging, cell fractionation, and gain/loss-of-function studies for ARF1.

Main Results:

  • Identified 215 RICTOR-proximal proteins, with 135 (63%) not previously linked to mTORC2 signaling.
  • Revealed approximately 30% of RICTOR resides in the nucleus, suggesting potential nuclear functions.
  • Identified ARF1 as an mTORC2-interacting protein and provided evidence for its negative regulatory role.

Conclusions:

  • Presented a novel method for studying endogenous mTORC2 and a comprehensive resource of its interactors.
  • Discovered potential nuclear roles for mTORC2 and identified ARF1 as a novel regulator of mTORC2 activity.
  • The findings offer new insights into the complex regulation and dynamics of mTORC2 signaling, particularly in response to insulin stimulation.
Keywords:
ADP ribosylation factorAkt/PKBBioID2RICTORcell signalinggrowth factormTORC2mammalian target of rapamycinproximity labeling

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