Figure 6. Palmitoylation was required for SCP1-mediated AKT inhibition.

(A) Palmitoylation was required for SCP1 to dephosphrylate AKT Ser473. HEK293T cells were transfected with WT-SCP1, DN-SCP1, and 2S-SCP1 for 24 h and treated with DMSO or 2-bromopalmitate (2BP; 10 μM) for 6 h. The phosphorylations of p-Ser473-AKT and p-Thr450-AKT were detected using western blotting. (B) Palmitoylation of SCP1 at C44 and C45 was required for its suppression of cell proliferation. Values represent mean ± SD (n = 3) (C) Palmitoylation inhibition increased the phosphorylation levels of endogenous AKT Ser473. HEK293T cells and H1299 cells were treated with DMSO or 2BP (10 μM) for 6 h. The phosphorylation of p-Ser473-AKT was detected using western blotting. (D) Depalmitoylation blocked the interaction between SCP1 and AKT. HEK293T cells were transfected with Myr-AKT-HA and FLAG-SCP1 or FLAG-SCP1-2S for 24 h and treated with DMSO or 2BP (10 μM) for 6 h. The interaction between AKT and SCP1 or SCP1-2S was detected using western blotting. (E) SCP1 blocked HUVEC migration in a palmitoylation-dependent manner. HUVECs were transfected with vector, WT-SCP1, DN-SCP1, and 2S-SCP1, respectively. Cell migration was detected using a wound healing assay. (F) SCP1 blocked HUVEC tube formation in a palmitoylation-dependent manner. HUVECs were transfected with vector, WT-SCP1, DN-SCP1, and 2S-SCP1, respectively. Tube formation was detected using a tube formation assay. (G) The working model for SCP1 palmitoylation and cell membrane localization is shown.

DOI: http://dx.doi.org/10.7554/eLife.22058.015

Figure 6—figure supplement 1. SCP1 suppresses AKT-mediated biological functions.

(A) The relative cell survival in Figure 6B was quantified. **p<0.01, *p<0.05. (B) The phosphatase activity of SCP1 was not affected by its palmitoylation status. The phosphatase activity was measured using a pNPP (p-nitrophenyl-phosphate) assay. (C) The migration was calculated, as shown in Figure 6E. **p<0.01, *p<0.05. (D) The tube length in Figure 6F was measured. **p<0.01, *p<0.05. (E) SCP1 impaired H1299 tumor bearing in nude mice in a palmitoylation-dependent manner. H1299 cells were infected with lentivirus with vector, WT-SCP1, DN-SCP1, or 2S-SCP1, and they were subcutaneously injected into nude mice (1 × 10⁷ per mouse). The tumor was photographed after 42 days. WT: wild-type.

DOI: http://dx.doi.org/10.7554/eLife.22058.016