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

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.