Figure 3. Regulation of mTORC1 by cytosolic calcium and CaM occurs proximal to mTORC1 itself.
(a) Effects of 10 μM CaM antagonist calmidazolium (CMDZ) or 25 μM cytosolic Ca2+ chelator BAPTA-AM (BAPTA) on the phosphorylation of different proteins of the mTOR signaling pathway at different time points. The bottom panel of plots shows the percentage of p-S6K and p-Akt (S473) levels compared with 0 min treated 293T cells normalized by total S6K and total Akt control, respectively (mean ± s.d., n = 3 independent experiments). (b and d) Effects of CMDZ (10 μM) or BAPTA-AM (25 μM) on the phosphorylation of indicated proteins in response to deprivation and stimulation with leucine (b) and insulin (d). Cell lysates were prepared from HEK293T cells deprived for 3 hr of leucine (b) or 24 hr of serum (d) and, where indicated, stimulated with 52 μg/ml leucine or 600 nM insulin for 10 min. CMDZ (10 μM) or BAPTA-AM (25 μM) was added 1 hr prior to cell harvesting. The plots show the percentage of p-S6K levels compared with vehicle control treated leucine (b) or serum (d) starved HEK293T cells normalized by total S6K control (mean ± s.d., n = 3 independent experiments, respectively). (c) Effects of CMDZ (10 μM) on the phosphorylation of S6K in HEK293T cells transfected with constitutively active RagA or RagC in expression vectors. Cell lysates were prepared and subjected to immunoblotting. The plot shows the percentage of p-S6K levels compared with vector transfected vehicle control treated 293T cells normalized by GAPDH loading control (mean ± s.d., n = 3 independent experiments). (e and f) Effects of 25 μM BAPTA-AM (e) and 10 μM CMDZ (f) on the phosphorylation state of S6K in HEK293T cells stably expressing constitutively active Rheb as indicated. HEK293T cells were transduced with lentiviral FLAG-tagged RhebN153T, and treated with indicated compounds for 1 hr. Cell lysates were prepared and used for immunoblotting. The plots show the percentage of p-S6K levels compared with vehicle control treated empty lentiviral vector transduced 293T cells normalized by total S6K control (mean ± s.d., n = 2 independent experiments). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. no significant difference.
Figure 3—figure supplement 1. Effects of calmodulin antagonist (a) calmidazolium (CMDZ), (b) W-7 and (c) cytosolic Ca2+ chelator BAPTA-AM on phosphorylation state of S6K (T389) and Akt (S473).
Figure 3—figure supplement 2. Effects of calmodulin antagonist calmidazolium (CMDZ) and cytosolic Ca2+ chelator BAPTA-AM on mTOR signaling pathway in HUVEC and A549 cells.
