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

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.