Figure 4. CaM interacts with mTORC1 and regulates mTORC1 kinase activity in vitro.

(a) CaM interacts with mTOR independent of hVps34 or raptor. Cell lysates were prepared from HEK293T cells transduced with lentiviral shRNAs targeting human mTOR, raptor, hVps34 or scrambled shRNA, followed by CaM sepharose precipitation in the presence of CaCl₂ (1 mM) or EGTA (5 mM). The cell lysates and precipitates were analyzed by immunoblotting to detect the indicated proteins. (b) Endogenous mTORC1 was pulled down by CaM sepharose in a Ca²⁺-dependent manner. HEK293T cells were lysed in CHAPS buffer, and the lysates were incubated with CaM sepharose in the presence of CaCl₂ (1 mM) or EGTA (5 mM). The precipitates were analyzed by immunoblotting. (c and d) Cell lysates were prepared from HEK293T cells in CHAPS buffer, and endogenous mTORC1 was immunoprecipitated by a raptor antibody. ATP (250 μM), Torin1 (100 nM), CMDZ (8 μM), CaM (2 μM) or/and CaCl₂ (1 mM) were added into the kinase reaction as indicated. Phosphorylation of 4EBP1 (c) and S6K (d) were detected by immunoblotting. The plots show the fold of change of phosphorylation of 4EBP1 (c) or S6K (d) compared with control group (first lane) normalized by total GST-tagged protein control. (mean ± s.d., n = 6 and 5 independent experiments, respectively).

DOI: http://dx.doi.org/10.7554/eLife.19360.010

Figure 4—figure supplement 1. Effects of calmidazolium (CMDZ) on hVps34 depleted cells.

(a) HEK 293T cells were transduced with lentivirus carrying scramble shRNA or shRNA targeting hVps34. 96 hr after puromycin selection, cells were treated with vehicle control or CMDZ (10 μM) for 1 hr. (b) The same cells in (a) were starved with amino acids for 1 hr, and re-stimulated with amino acids for 10 min. Cells were lysed and subjected to immunoblotting.

DOI: http://dx.doi.org/10.7554/eLife.19360.011

Figure 4—figure supplement 2. CaM interacts with mTORC1 independently of raptor or hVps34.

(a) The interaction between CaM and mTORC1 is sensitive to detergents. Cell lysates were prepared from HEK293T cells in lysis buffer containing 1% Triton X-100 or 1% NP-40. Endogenous mTORC1 was precipitated by CaM sepharose in the presence of Ca²⁺ or EGTA. (b) The interaction between CaM and mTOR is inhibited by the CaM antagonist W-7. Cell lysates were prepared from HEK293T cells in CHAPS buffer, and endogenous mTOR was precipitated by CaM sepharose in the presence of W-7 or vehicle control.

DOI: http://dx.doi.org/10.7554/eLife.19360.012

Figure 4—figure supplement 3. The presence or absence of Ca²⁺ does not affect the association of mTORC1.

Cell lysates were prepared from HEK293T in CHAPS buffer, and endogenous mTORC1 was immunoprecipitated by mTOR antibody. Rabbit IgG was used as mock immunoprecipitation.

DOI: http://dx.doi.org/10.7554/eLife.19360.013

Figure 4—figure supplement 4. Proposed model of regulation of mTORC1 by TRPML1, lysosomal calcium and CaM.

Upon the translocation of mTORC1 onto the lysosome through binding to GTP-Rheb, lysosomal Ca²⁺ released through TRPML1 enriches the local Ca²⁺ concentration, prompting the binding to and activation of CaM, which in turn interacts with mTORC1 and leading to a fully activation of the kinase activity.

DOI: http://dx.doi.org/10.7554/eLife.19360.014