Fig. 6
SirT6 regulates nuclear levels of SKP2 through deacetylation. a Analysis of the effect of SirT6 on the SKP2 levels. 293F and HeLa cells were co-transfected with the indicated combinations of FLAG-SKP2 and SirT6-HA. Tubulin was included as loading control. b Levels of acetylation in FLAG-SKP2 purified from 293F cells downregulated in SirT6 (shScramble vs shSirT6, left) or overexpressing SirT6-HA in 293F cells expressing shRNA SirT6 (right). SKP2 was purified with FLAG resin and analyzed by western-blot with anti-acetyl-lysine antibodies. c Posttranslational modifications in SKP2 in the presence or absence of SirT6. Upper panel: Summary of the procedure. Lower panel: Summary of the identified PTMs in SKP2 in the indicated conditions. Additional data is shown in Supplementary Data Set 2. d Left, ESI-MS spectrum of acetylated peptide (aa 72–83, 72K.SK73acGSDK77acDFVIVR.R.83-C) from SKP2 − / + SirT6 of a representive experiment of two replicas. Signal was detected at m/z 464.92 (charge state 4). Middle and right, ESI-MS spectrum of phosphorylated peptides (aa 72–83, 72K.S72phKGS75phDKDFVIVR.R.83-C, 72K.KGS75phDKDFVIVR.R.83-C) from SKP2 − / + SirT6. Signal appeared at m/z 477.61 and 477.61, respectively). e Analysis of the ability of SirT6-HA to upregulate the levels of nuclear FLAG-tagged SKP2, as either WT or tetramutated in K68R/K71R/K73R/K77R. Western blotting of nuclear extracts from 293F cells previously transfected with the indicated constructs. f Quantification of n = 5 experiments as the one showed in e. SKP2 levels were normalized with histone H3. All the values were represented relative to the normalized levels of WT SKP2 in the absence of SirT6 (T-test; s.e.m., **:p < 0.01). g Same experiment as in c, d, but testing the levels of nuclear SKP2 double mutant S72D/S75D instead of the tetramutant. h Quantification of n = 6 experiments as in g and represented as in f (T-test; SEM, *p < 0.05, ***p < 0.005)
