Figure 6. SIRT1 overexpression blunts MAP4K3 amino acid-dependent activation of mTORC1 and deletion mapping of SIRT1 interaction with MAP4K3.

(A) We transfected WT HEK293A cells with either SIRT1-HA or pcDNA empty vector and maintained the cells under baseline complete media (CM) conditions or subjected the cells to amino acid starvation (−AA) or amino acid starvation followed by amino acid restimulation (+AA). 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 suppression of mTORC1 activation in WT cells overexpressing SIRT1. α-tubulin served as the loading control. (A, B) TOP GRAPH: we quantified the levels of phosphorylated S6 and total S6 in (A) by densitometry, determined the ratio of phosphorylated S6: total S6, and normalized the results to pcDNA-transfected WT HEK293A cells at the baseline (arrow in (A)). **P < 0.01, ANOVA with post-hoc Tukey test. (A) BOTTOM GRAPH: we quantified the levels of phosphorylated 4E-BP1 and total 4E-BP1 in (A) by densitometry, determined the ratio of phosphorylated 4E-BP1: total 4E-BP1, and normalized the results to pcDNA-transfected WT HEK293A cells at the baseline (arrow in (A)). **P < 0.01, ANOVA with post-hoc Tukey test; n = 4 biological replicates. Error bars = s.e.m. (C) Diagram of SIRT1 deletion constructs used for mapping its interaction with MAP4K3. LEFT: full-length SIRT1 with enzymatic deacetylase domain indicated. RIGHT: diagrams of the different SIRT1 deletion constructs used in the co-transfection, co-immunoprecipitation studies. (D) We co-transfected HEK293A cells with either WT-MAP4K3-FLAG or kinase dead (KD)-MAP4K3-FLAG and with a different SIRT1-HA deletion construct, as indicated. We then performed co-immunoprecipitation of MAP4K3 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 on the left.