Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

The mechanistic target of rapamycin complex 1 (mTORC1) functions as a master regulator of cell growth and proliferation. We previously demonstrated that intracellular calcium ion (Ca²⁺) concentration modulates the mTORC1 pathway via binding of the Ca²⁺ sensor protein calmodulin (CaM) to tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTORC1. However, the precise molecular mechanism by which Ca²⁺/CaM modulates mTORC1 activity remains unclear. Here, we performed a binding assay based on nano-luciferase reconstitution, a method for detecting weak interactions between TSC2 and its target, Ras homolog enriched in the brain (Rheb), an activator of mTORC1. CaM inhibited the binding of TSC2 to Rheb in a Ca²⁺-dependent manner. Live-cell imaging analysis indicated increased interaction between the CaM-binding region of TSC2 and CaM in response to elevated intracellular Ca²⁺ levels. Furthermore, treatment with carbachol, an acetylcholine analog, elevated intracellular Ca²⁺ levels and activated mTORC1. Notably, carbachol-induced activation of mTORC1 was inhibited by CaM inhibitors, corroborating the role of Ca²⁺/CaM in promoting the mTORC1 pathway. Consistent with the effect of Ca²⁺/CaM on the TSC2-Rheb interaction, increased intracellular Ca²⁺ concentration promoted the dissociation of TSC2 from lysosomes without affecting Akt-dependent phosphorylation of TSC2, suggesting that the regulatory mechanism of TSC2 by Ca²⁺/CaM is distinct from the previously established action mechanism of TSC2. Collectively, our findings offer mechanistic insights into TSC2-Rheb regulation mediated by Ca²⁺/CaM, which links Ca²⁺ signaling to mTORC1 activation.