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. 2023 May 23;6(8):e202201525. doi: 10.26508/lsa.202201525

Figure 2. Activated MAP4K3 represses AMPK to turn on the mTORC1 complex.

Figure 2.

(A) WT and MAP4K3 k.o. cells were starved of amino acids for 3 h (−) or starved of amino acids for 3 h and then restimulated with amino acids for 10 min (+). We immunoblotted the resulting cell protein lysates for phosphorylated Akt, total Akt, phosphorylated S6 kinase 1, phosphorylated S6, and phosphorylated 4E-BP1 as indicated. β-actin served as the loading control. (B) WT cells and two independently derived lines (two different guide RNAs; k.o. 1 = M1, k.o. 2 = M4) of MAP4K3 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 AMPK α1 subunit, total AMPK α1 subunit, and MAP4K3 as indicated. β-actin served as the loading control, and we did not detect any changes in adenine nucleotide levels between WT and MAP4K3 k.o. cells. (C) WT cells, MAP4K3 k.o. cells (M1), AMPK α1 k.o. cells, and MAPK3 (M1)/AMPK α1 double k.o. cells were starved of amino acids for 3 h (−) or starved of amino acids for 3 h and then restimulated with amino acids for 10 min (+). We immunoblotted the resulting cell protein lysates for phosphorylated Akt, total Akt, phosphorylated ACC, phosphorylated AMPK, phosphorylated S6 kinase 1, phosphorylated S6, and phosphorylated 4E-BP1. Note complete rescue of mTORC1 activation in the MAP4K3/AMPK α1 double k.o. cell line. β-actin served as the loading control. (D) We cultured a WT HEK293A cell line, MAP4K3 k.o. cell line, AMPK α1 k.o. cell line, and MAPK3/AMPK α1 double k.o. cell line in complete media over 72 h. Here, we see the quantification of cell numbers at 24 h intervals. ***P < 0.001; ANOVA with post-hoc Tukey test; n = 3 technical replicates. See Fig S4 for bar graph of terminal cell number data. Error bars = s.e.m.