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 (Ca) concentration modulates the mTORC1 pathway via binding of the Ca sensor protein calmodulin (CaM) to tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTORC1. However, the precise molecular mechanism by which Ca/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 Ca-dependent manner. Live-cell imaging analysis indicated increased interaction between the CaM-binding region of TSC2 and CaM in response to elevated intracellular Ca levels. Furthermore, treatment with carbachol, an acetylcholine analog, elevated intracellular Ca levels and activated mTORC1. Notably, carbachol-induced activation of mTORC1 was inhibited by CaM inhibitors, corroborating the role of Ca/CaM in promoting the mTORC1 pathway. Consistent with the effect of Ca/CaM on the TSC2-Rheb interaction, increased intracellular Ca concentration promoted the dissociation of TSC2 from lysosomes without affecting Akt-dependent phosphorylation of TSC2, suggesting that the regulatory mechanism of TSC2 by Ca/CaM is distinct from the previously established action mechanism of TSC2. Collectively, our findings offer mechanistic insights into TSC2-Rheb regulation mediated by Ca/CaM, which links Ca signaling to mTORC1 activation.