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

Figure 4. MAP4K3 activation of mTORC1 is partially mediated via Rheb.

Figure 4.

(A) WT HEK293A cells and two independently derived MAP4K3 HEK293A k.o. cell lines (M1 = k.o. 1 and M4 = k.o. 2) were cultured in complete media (+) or in low-glucose, amino acid-free media (−), as indicated, for 3 h. Protein lysates were then incubated with GTP-binding beads, and the immobilized proteins were immunoblotted for Rheb. (B) Protein lysates were prepared from a WT HEK293A cell line and two independently generated MAP4K3 k.o. cell lines (M1 and M4) cultured in complete media and immunoprecipitated with anti-Rheb antibody for endogenous TSC2. Note the decreased interaction of Rheb with TSC2 in MAP4K3 k.o. cell lines. (C) WT HEK293A cells and two independently derived MAP4K3 HEK293A k.o. cell lines (M1 and M4) were starved of amino acids for 3 h and restimulated with amino acids for 10 min. We then performed co-immunoprecipitation of Raptor and Rheb by Myc IP, followed by immunoblotting with anti-HA antibody or anti-Myc antibody. Immunoblotting of protein lysates from input cells is shown on the left. (D) WT and MAP4K3 k.o. cells were transfected with constitutively active Rheb as indicated, and starved of amino acids for 3 h (−) or starved of amino acids for 3 h and then restimulated with amino acids for 10 min (+). Protein lysates were prepared and immunoblotted for the indicated proteins. β-actin served as the loading control.