Figure 3. MAP4K3 amino acid dependent activation of mTORC1 occurs via LKB1 repression.

(A) WT and MAP4K3 k.o. cells (M1 line) were transfected with an LKB1-FLAG vector and then starved of amino acids for 3 h (amino acid depleted) or starved of amino acids for 3 h and restimulated with amino acids for 10 min (amino acid re-fed). The cells were fixed, immunostained with anti-FLAG antibody (green), and counterstained with DAPI (blue). Scale bar = 10 μm. (A, B) Quantification of the percentage of cells showing LKB1 localization to the cytosol in (A). Note that LKB1 is not retained in the nucleus in MAP4K3 k.o. cells upon amino acid stimulation. P < 0.01, two-tailed t test; n = 3 biological replicates. Error bars = s.e.m. (C) WT and MAP4K3 k.o. cells (M1 line) were cultured in complete media (CM), starved of amino acids for 3 h (−AA) or starved of amino acids for 3 h, and then restimulated with amino acids for 10 min (+AA). We performed subcellular fractionations and immunoblotted the resultant protein lysates for LKB1, Lamp1 or histone H2B, as indicated, quantified cytosolic LKB1 by densitometry, and normalized the results to the level of cytosolic LKB1 in amino acid-starved WT cells, which was arbitrarily set to 1. *P < 0.05, ANOVA with post-hoc Tukey test. See Fig S5A for longer exposure of LKB1 to permit visualization of nuclear LKB1. (D) WT cells, MAP4K3 k.o. cells (M1), LKB1 k.o. cells, and MAPK3 (M1)/LKB1 double k.o. cells were starved of amino acids for 3 h and then restimulated with amino acids for 30 min. We immunoblotted the resulting cell protein lysates for phosphorylated S6 kinase 1, phosphorylated S6, phosphorylated 4E-BP1, phosphorylated AMPK α1 subunit, and LKB1, as indicated. Note the complete rescue of mTORC1 activation in the MAP4K3/LKB1 double k.o. cell line. β-actin served as the loading control.