The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity
J. Clin. Invest. Jun Nakae, et al. 116:2473 doi:10.1172/JCI25518 [Go to this article.]

Figure 5
Sirt1 interacts with 3A FoxO1 but not with 3A/LXXAA FoxO1, and disruption of the LXXLL motif enhances acetylation of FoxO1. (A) SV40-transformed hepatocytes were transduced with adenovirus encoding Flag 3A FoxO1 (lane 1) or Flag 3A/LXXAA FoxO1 (lane 2). After 24 hours serum starvation, cells were cultured in AMEM supplemented with 0.1% bovine serum albumin, 0.5 mM 8-Br-cAMP, 0.5 mM IBMX, and 1 μM dexamethasone for 8 hours. Lysates were immunoprecipitated using anti-Flag monoclonal antibody (M2) and Western blotted with anti-Sir2 polyclonal (first panel) or anti-Flag monoclonal antibody (M2) (fourth panel) or immunoprecipitated using anti-Sir2 polyclonal antibody and Western blotted with anti-Sir2 antibody (second panel) or anti-Flag monoclonal antibody (third panel). (B) SV40-transformed hepatocytes transduced with adenovirus encoding Flag 3A FoxO1 (lane 1) or Flag 3A/LXXAA FoxO1 (lane 2) were incubated with 0.5 mM 8-Br-cAMP, 0.5 mM IBMX, and 1 μM dexamethasone for 8 hours after 24 hours of serum starvation. Lysates were immunoprecipitated using anti-Flag monoclonal antibody (M2) and Western blotted with anti-acetylated lysine polyclonal (top panel) or anti-Flag monoclonal antibody (M2) (bottom panel). (C) Quantification of the data in B. Mean ± SEM of the folds of acetylation of 3A/LXXAA was calculated from 3 independent experiments using NIH Image 1.62 (http://rsb.info.nih.gov/nih-image/). Subsequent blotting with anti-Flag antibody using the same filter normalized acetylation of each FoxO1. (D) HEK293 cells transfected with pCMV5/cMyc/3A FoxO1 (lanes 1–4) or 3A/LXXAA FoxO1 (lanes 5–8) were incubated for 8 hours in the absence or presence of nicotinamide (50 mM) or trichostatin A (TSA) (2 μM). cMyc-tagged FoxO1 was immunoprecipitated, and the acetylation of FoxO1 was assessed by Western blot with the antibody to acetylated lysine (upper panel). Total levels of FoxO1 were assessed with the antibody to cMyc (lower panel).