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

Figure 7. Phosphorylation status of threonine 344 in SIRT1 regulates amino acid-dependent activation of mTORC1.

Figure 7.

(A) We transfected WT HEK293A cells with SIRT1-HA or pcDNA empty vector, and transfected MAP4K3 HEK293A k.o. cells (M4 line) with SIRT1-HA, SIRT1-T344D phosphomimetic mutant or pcDNA empty vector, and maintained cells under baseline complete media (CM) conditions or subjected cells to amino acid starvation (−) or amino acid starvation followed by amino acid restimulation (+). We immunoblotted the resulting cell protein lysates for MAP4K3, HA-tagged SIRT1, phosphorylated S6, total S6, phosphorylated 4E-BP1, and total 4E-BP1, as indicated. Note the rescue of mTORC1 activation in MAP4K3 k.o. cells expressing the SIRT1-T344D mutant in comparison with MAP4K3 k.o. cells expressing pcDNA empty vector or SIRT1-HA (boxed). α-tubulin served as the loading control. (A, B) LEFT: we quantified phosphorylated S6 and total S6 in MAP4K3 k.o. cells in (A) by densitometry, determined the ratio of phosphorylated S6: total S6, and normalized the results to pcDNA-transfected WT HEK293A cells in complete media (arrow in (A)). **P < 0.01, ANOVA with post-hoc Tukey test. (A) RIGHT: we quantified phosphorylated 4E-BP1 and total 4E-BP1 in MAP4K3 k.o. cells in (A) by densitometry, determined the ratio of phosphorylated 4E-BP1: total 4E-BP1, and normalized the results to pcDNA-transfected WT HEK293A cells in complete media (arrow in (A)). *P < 0.05, ANOVA with post-hoc Tukey test; n = 3 biological replicates. Error bars = s.e.m. (C) We transfected WT HEK293A cells with FLAG-LKB1 alone or in combination with WT SIRT1-HA or with pcDNA empty vector, and transfected MAP4K3 HEK293A k.o. cells (M4 line) with FLAG-LKB1 alone, FLAG-LKB1 and WT SIRT1-HA or FLAG-LKB1 and SIRT1-HA-T344D. We then performed co-immunoprecipitation of LKB1 and SIRT1 by FLAG IP, followed by immunoblotting with anti-HA antibody or anti-FLAG antibody. Immunoblotting of protein lysates from input cells is shown below. (C, D) Quantification of SIRT1–LKB1 interaction in (C) based upon densitometry analysis performed on WT HEK293A cells co-transfected with FLAG-LKB1 and WT-SIRT1-HA, MAP4K3 k.o. cells co-transfected with FLAG-LKB1 and WT SIRT1-HA, and MAP4K3 k.o. cells co-transfected with FLAG-LKB1 and SIRT1-HA-T344D. Results for SIRT1: LKB1 were normalized to WT HEK293A cells co-transfected with FLG-LKB1 and WT-SIRT1-HA (arrow in (C)). P < 0.01, two-tailed t test; n = 3 biological replicates. Error bars = s.e.m.