KAT7-mediated CANX (calnexin) crotonylation regulates leucine-stimulated MTORC1 activity

Autophagy. 2022 Dec;18(12):2799-2816. doi: 10.1080/15548627.2022.2047481. Epub 2022 Mar 10.

Abstract

Amino acids play crucial roles in the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) pathway. However, the underlying mechanisms are not fully understood. Here, we establish a cell-free system to mimic the activation of MTORC1, by which we identify CANX (calnexin) as an essential regulator for leucine-stimulated MTORC1 pathway. CANX translocates to lysosomes after leucine deprivation, and its loss of function renders either the MTORC1 activity or the lysosomal translocation of MTOR insensitive to leucine deprivation. We further find that CANX binds to LAMP2 (lysosomal associated membrane protein 2), and LAMP2 is required for leucine deprivation-induced CANX interaction with the Ragulator to inhibit Ragulator activity toward RRAG GTPases. Moreover, leucine deprivation promotes the lysine (K) 525 crotonylation of CANX, which is another essential condition for the lysosomal translocation of CANX. Finally, we find that KAT7 (lysine acetyltransferase 7) mediates the K525 crotonylation of CANX. Loss of KAT7 renders the MTORC1 insensitivity to leucine deprivation. Our findings provide new insights for the regulatory mechanism of the leucine-stimulated MTORC1 pathway.Abbreviations: CALR: calreticulin; CANX: calnexin; CLF: crude lysosome fraction; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; GST: glutathione S-transferase; HA: hemagglutinin; HEK293T: human embryonic kidney-293T; KAT7: lysine acetyltransferase 7; Kcr; lysine crotonylation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LAMTOR/Ragulator: late endosomal/lysosomal adaptor: MAPK and MTOR activator; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PDI: protein disulfide isomerase; PTM: post-translational modification; RPS6KB1/p70S6 kinase 1: ribosomal protein S6 kinase B1; RPTOR: regulatory associated protein of MTOR complex 1; SESN2: sestrin 2; TMEM192: transmembrane protein 192; ULK1: unc-51 like autophagy activating kinase 1.

Keywords: CANX; KAT7; LAMP2; MTORC1; leucine; lysine crotonylation; ragulator.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy*
  • Calnexin* / metabolism
  • HEK293 Cells
  • Humans
  • Leucine / metabolism
  • Leucine / pharmacology
  • Lysine / metabolism
  • Lysine Acetyltransferases* / metabolism
  • Lysosomal-Associated Membrane Protein 2 / metabolism
  • Lysosomes / metabolism
  • Mechanistic Target of Rapamycin Complex 1* / metabolism
  • Signal Transduction

Substances

  • Calnexin
  • KAT7 protein, human
  • Leucine
  • Lysine
  • Lysine Acetyltransferases
  • Lysosomal-Associated Membrane Protein 2
  • Mechanistic Target of Rapamycin Complex 1
  • CANX protein, human

Grants and funding

This work was supported by the National Natural Science Foundation of China (31520103915), the National Key Basic Research Program of China (973 Program) (National Basic Research Program of China (973 Program) 2013CB127305), Hubei Provincial Natural Science Foundation of China (Natural Science Foundation of Hubei Province 2018CFA020), and the Fundamental Research Funds for the Central Universities (2013PY056, 2662015PY111, and 2013JQ001).