FIGURE 6.
Ca2+ and CaMKK-β are also implicated in mTORC1 inactivation by amino acid starvation. A, NIH3T3 cells were grown for 2 h in KH plus AA with (+CaCl2) or without CaCl2 and supplemented with 100 μm EGTA (−CaCl2) in the presence or absence of 10 μm BAPTA-AM. When indicated, rapamycin (100 nm) was used during the last hour as a control. In the last 30 min of incubation, AA were removed or not as indicated. Cell extracts (75 μg of protein) were analyzed by immunoblot using anti-p70S6K, anti-P-p70S6K (Thr-389), anti-ULK1, anti-P-ULK1 (Ser-757), and as a loading control, anti-actin antibodies. B, fura-2AM-loaded NIH3T3 cells incubated for 1 h with rapamycin (100 nm) were imaged as described under “Experimental Procedures.” The effect of two rounds of AA addition and withdrawal on [Ca2+]c was measured in cells incubated in KH without CaCl2 and containing 100 μm EGTA. The mean values and S.D. at each time point of more than 50 cells from two different cultures are shown. No significant differences were observed in the effects of addition/withdrawal of AA on [Ca2+]c with cells not treated with rapamycin. Ionomycin (1 μm) was used as a positive control. C, NIH3T3 cells were previously incubated for 2 h in KH supplemented with AA with or without BAPTA-AM (10 μm). When indicated, rapamycin (100 nm) was used during the last hour as a control. Subsequently, AA were removed (KH) or not (AA) as indicated, and cells were loaded with 5 μm fluo-3AM for 30 min. The fluorescence intensity of fluo-3AM in the cells was measured by flow cytometry. Values are the means and S.D. from two separate experiments with quadruplicate samples. The fluorescence emitted by 10,000 cells in each case is showed below. D, NIH3T3 cells were grown and treated as described in the legend to Fig. 2B. Cell extracts (75 μg of protein) were analyzed by immunoblot using anti-p70S6K, anti-P-p70S6K (Thr-389), and as a loading control, anti-actin antibodies. E, NIH3T3 cells were transfected with CaMKK-β (Si CaMKK-β) or with negative control (C-) siRNAs. After 72 h, cells were incubated as above and analyzed by immunoblot using the same antibodies as in A plus anti-CaMKK-β. Densitometric measurements of the ratio of P-p70S6K to total p70S6K from four independent experiments in each case are also shown on the right of D and E. In E, low and high exposure (exp.) blots are shown. Differences were found to be statistically significant at p < 0.0005 (***) and p < 0.005 (**). NS, no significant differences.