(D) 2 105 Lewis lung carcinoma?cells were injected into SCP1-wild-type or -knockdown littermates. AKT-S473D. The cells were placed in plates coated with Matrigel and tubular structures were photographed after 6 h. The tube lengths AMG-925 were measured in each field. (B) HUVECs were transfected with siSCP1 and treated with or without AKT inhibitor (AKTi; MK2206, 2 nM) for 5 days as indicated. The tube lengths were measured in each field. (C) Cell migration was detected using a wound healing assay. HUVECs were transfected and treated with or without AKTi (MK2206, 2 nM). The migration cell number in each field was calculated.DOI: http://dx.doi.org/10.7554/eLife.22058.011 elife-22058-fig4-data1.xlsx (9.5K) DOI:?10.7554/eLife.22058.011 Abstract SCP1 as a nuclear transcriptional regulator acts globally to silence neuronal genes and to affect the dephosphorylation of RNA Pol ll. However, we report the first finding and description of SCP1 as a plasma membrane-localized protein in various cancer cells using EGFP- or other epitope-fused SCP1. Membrane-located SCP1 dephosphorylates AKT at serine 473, leading to the abolishment of serine 473 phosphorylation that results in suppressed angiogenesis and a decreased risk of tumorigenesis. Consistently, we observed increased AKT phosphorylation and angiogenesis followed by enhanced tumorigenesis in (which encodes SCP1) gene – knockout mice. Importantly, we discovered that the membrane localization of SCP1 is crucial for impeding angiogenesis and tumor growth, and this localization depends on palmitoylation of a conserved cysteine motif within its NH2 terminus. Thus, our study discovers a novel mechanism underlying SCP1 shuttling between the plasma membrane and nucleus, which constitutes AMG-925 a unique pathway in transducing AKT signaling that is closely linked to angiogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.22058.001 to are?shown. (D) HeLa cells were transfected with WT-SCP1, C44S-SCP1, C45S-SCP1, C47S-SCP1, and C44/45S(2S)-SCP1 for 24 h. The subcellular localizations of WT-SCP1 and its mutants were detected using immunofluorescence assay. (E) HEK293T cells were transfected with WT-SCP1, C44S-SCP1, C45S-SCP1, and C44/45S(2S)-SCP1 for 24 h and cell fractions of were analyzed using western blotting. (F) FLAG-SCP1 was expressed in HEK293T cells, immunoprecipitated, and palmitoylation was detected using the?acylCbiotin exchange?(ABE) assay. (G) Palmitoylation of endogenous SCP1 in HEK293T cells was detected using the?ABE assay. (H) FLAG-SCP1 was expressed in HEK293T cells for 24 h and treated with 2BP (10 M) or palmostatin B (50 IFRD2 M) for 12 h. Palmitoylation of SCP1 was detected using pan-palmitoylation antibody. (I) and (J) Identification of palmitoylation sites using the?ABE assay (I) and the?[3H] palmitate incorporation assay (J). DOI: http://dx.doi.org/10.7554/eLife.22058.005 Figure 2figure supplement 1. Open in a separate window SCP1 membrane localization depends on its palmitoylation.(A) The membrane localization of SCP1 was not affected by farnesyltransferase or prenyltransferase inhibitor.?The transfected HeLa cells were treated with AMG-925 DMSO, FTI-277(10 M), or GGTI (15 M) for 8 h. (B) The membrane localization of SCP1 was blocked by palmitoyltransferase inhibitor. GFP-Ras and/or GFP-SCP1 were co-expressed AMG-925 in HeLa cells for 24 h. The transfected cells were treated with DMSO or 2BP?(10 M) for 8 h. (C) The membrane localizations of SCP2 and SCP3 were blocked by palmitoyltransferase inhibitor. HeLa cells were transfected with GFP-SCP2/SCP3 for 24 h. (D) The newly synthesized SCP1 was transported to the?Golgi without palmitoylation and then translocated to the plasma membrane by palmitoylation. HeLa cells were transfected with GFP-Golgi and mCherry-SCP1 for 24 h and treated with 2BP?(10 M) or cycloheximide?(15 g/ml) for 8 h. (E) The working model for SCP1 palmitoylation and cell membrane location?is?shown. (F) Amino acid residues from 31 to 55 are important for SCP1 palmitoylation and cell membrane localization. HeLa cells were transfected with the truncated mutant of GFP-SCP1 31C55 for 24 h and treated with DMSO or 2BP?(10 M) for 8 h. DOI: http://dx.doi.org/10.7554/eLife.22058.006 It has been reported that palmitoylated proteins can be recycled from the?plasma membrane to the?Golgi (Resh, 2006). Therefore, we tested whether the nucleus- or Golgi-localized SCP1 was newly synthesized and recycled to the?nucleus or Golgi from the?plasma membrane. SCP1 localization was monitored in transfected HeLa cells treated with cycloheximide (CHX) for 8 h to block new protein synthesis.