Figure 5. MAP4K3 interacts with and phosphorylates SIRT1.

(A) HEK293A cells were transfected with SIRT1-HA and WT-MAP4K3-FLAG, kinase dead (KD)-MAP4K3-FLAG, FLAG-GFP empty vector (negative control), or FLAG-LKB1 (positive control). After performing immunoprecipitation (IP) with anti-FLAG antibody, we immunoblotted the IP material for the indicated proteins. (B) We incubated in vitro transcribed–translated SIRT1-HA with anti-FLAG IP material isolated from cells expressing WT-MAP4K3-FLAG, KD-MAP4K3-FLAG or FLAG-GFP empty vectors. We then performed FLAG pull-downs, and immunoblotted with anti-HA antibody and anti-FLAG antibody, which confirmed a physical interaction between recombinant SIRT1 and MAP4K3. (C) We transfected WT or MAP4K3 k.o. HEK293A cells with SIRT1-HA, allowed them to grow in labelling media with P³² orthophosphate, and subjected the cells to amino acid starvation (−AA) or amino acid starvation followed by amino acid restimulation (+AA). SIRT1 was immunoprecipitated, digested with glutamyl endopeptidase, and the resulting peptide mix was spotted on cellulose thin-layer plates for separation by electrophoresis followed by ascending chromatography and then autoradiography to visualize phosphopeptides. Circles indicate MAP4K3-dependent phosphorylated peptide fragments of SIRT1. (C, D) After isolating SIRT1 as in (C), SIRT1 was acid hydrolyzed, and after mixing with unlabeled phosphoamino acid standards, the amino acid mixtures were separated by two-dimensional electrophoresis on thin-layer chromatography plates followed by autoradiography to visualize the P³²-labeled phosphoamino acids. Unlabeled phosphoamino acid standards were visualized by spraying the thin-layer chromatography plates with ninhydrin and autoradiography films were aligned with the plates to identify the P³²-labeled phosphoamino acids from the SIRT1 samples. S = phosphoserine and T = phosphothreonine. Products of partial hydrolysis are circled.