Figure 6: AKT phosphorylates FAF1 at Ser 582 and inhibits its membrane localization.

(a) Sequence alignment of the AKT phosphorylation site within FAF1 orthologs from different species and the known AKT-phosphorylating proteins p27, p21 and Skp2. (b) AKT-phosphorylated FAF1 peptide identified by mass spectrometry analysis. (c) Immunoblot (IB) of total cell lysate (TCL) and immunoprecipitants derived from HeLa cells transfected with Myr-HA-AKT and treated with increasing doses of LY294002 (25 and 50 μM) for 12 h as indicated. NS, non-specific antibody. (d) HEK293T cells were transfected with Flag-FAF1 wt or S582A, T426A, S270A or S320A mutants along with Myr-HA-AKT expression plasmids as indicated. Cells were then collected for immunoprecipitation (IP) and IB analysis. (e) IB analysis of TCL and anti-FAF1 immunoprecipitates derived from serum-starved HeLa cells treated with IGF-1 (200 ng ml⁻¹), TGF-β (5 ng ml⁻¹) and LY294002 (50 μM) as indicated for 8 h. (f) HeLa cells stably overexpressing FAF1 wt or FAF1 S582A mutant constructs were transfected with or without HA-Myr-AKT as indicated. Cells were then collected for membrane (Mem), cytoplasm (Cyto) and nuclear (Nuc) extraction and IB analysis. (g) IB of Mem, Cyto and Nuc extraction derived from HeLa cells transfected with or without HA-Myr-Akt and treated with LY294002 (50 μM for 12 h) as indicated. (h) HeLa cells were treated with TGF-β (5 ng ml⁻¹) and selective PI3K inhibitors LY294002 (50 μM) or BKM120 (1 μM) as indicated for 8 h. Cells were then collected for Mem, Cyto and Nuc extraction and IB analysis. (i) Hypothetical working model of AKT-mediated FAF1 phosphorylation: phosphorylated FAF1 does not efficiently attach to the cell surface or associate with the VCP/E3 complex.