Gal3‐CaN‐Smurf1 Complex Sequestrates FLCN‐FNIPs to Facilitate TFEB Activation in Response to Endomembrane Damage

Abstract Smurf1 mediates lysosomal biogenesis upon endomembrane damage by interacting with lysosomal injury sensor Gal3 and phosphatase CaN to form Gal3‐CaN‐Smurf1 complex, which is critical for TFEB dephosphorylation. However, whether Smurf1 plays a role in the inhibition of mTOR‐mediated TFEB phosphorylation is still unclear. TFEB phosphorylation by mTORC1 is strictly dependent on RagC/D GTPase activating protein FLCN. Here, we found that Smurf1 promotes the dissociation of RagC from TFEB upon lysosomal damage, selectively impairing TFEB phosphorylation. These findings suggest that the lysosomal damage‐induced Gal3‐CaN‐Smurf1 complex sequesters FLCN‐FNIPs to facilitate TFEB activation. This disruption of FLCN GAP function toward RagC/D impairs TFEB's lysosomal localization and phosphorylation. Notably, FLCN K462R and/or FNIP2 K466R mutations reduce their binding affinity with the Gal3‐CaN‐Smurf1 complex, suggesting Smurf1‐mediated poly‐ubiquitylation of FLCN K462 and FNIP2 K466 plays a role for pentamer formation. Indeed, sequestration of FLCN‐FNIPs stabilizes the Gal3‐CaN‐Smurf1 complex, wherein Smurf1 directly binds and ubiquitinates TFEB. This facilitates TFEB's dephosphorylation and activation. These findings indicate that Gal3‐CaN‐Smurf1 complex interconnects with the FLCN‐FNIPs to orchestrate TFEB localization and activity in response to lysosomal damage stress. Understanding Smurf1's regulation in the mTOR‐TFEB axis, which balances tumor growth and stress‐induced cell homeostasis, may provide novel therapeutic targets for tumor progression and drug resistance.