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  • br Figure The PDSST Motif in

    2020-08-12


    Figure 3. The 66PDSST70 Motif in NANOG Is the Degron Recognized by SPOP (A) Sequence alignment of NANOG with the SPOP binding motif (SBC) among different species.
    (B) FLAG-SPOP and indicated HA-tag NANOG plasmids were co-expressed in HEK293T sybr安全dna凝胶染料 (3A: S68A, S69A, T70A). Cell lysates were prepared for coIP and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting.
    (C) GST and GST-SPOP proteins were purified from E. coli and incubated with NANOG or PDSST deletion protein IP from HEK293T cells. The products of a pull-down assay were analyzed by WB.
    (D) FLAG-NANOG, HA-SPOP, and His-ubiquitin were co-expressed in HEK293T cells. After treatment with MG132 (10 mM) for 6 hr, the Ni-NTA ubiquitination assay was performed and analyzed by WB.
    (E) FLAG-NANOG or PDSST deletion mutant was co-expressed in HEK293T cells with HA-SPOP. After treating cells with CHX (10 mg/mL) for indicated time intervals, protein levels of NANOG and SPOP were analyzed by WB.
    (F) The NANOG protein abundance in (E) was quantified by ImageJ and plotted as indicated.
    (G) HA-SPOP and indicated FLAG-tag NANOG plasmids were co-expressed in HEK293T cells. Cell lysates were prepared for coIP and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting.
    (H) Ectopically expressed SPOP promotes protein degradation of exogenous WT-NANOG but not D67A or S68A mutants. 293T cells were transfected with indicated constructs for 24 hr followed by WB.
    (I) FLAG-NANOG or mutants were co-expressed in HEK293T cells with HA-SPOP. After treating cells with CHX (10 mg/mL) for indicated time intervals, protein levels of NANOG and SPOP were analyzed by WB.
    (J) The NANOG protein abundance in (I) was quantified by ImageJ and plotted as indicated.
    (K) Binding was examined using biotinylated peptide pull-down assay and analyzed using WB.
    In contrast, co-expression of SPOP dramatically reduced the sphere-formation activity of NANOG-WT, but not that of the NANOG S68Y mutant (Figures 4H and 4I). In addition, SPOP inhibited the effect of WT-NANOG but not the NANOG SBC-deleted or S68Y mutants on migration and proliferation of PCa cells (Figures S4D–S4F). Subcutaneous injection of DU145 cells expressing the NANOG S68Y mutant into nude mice promoted tumor growth even under the condition of SPOP overexpression (Figures 4J and 4K). Together, these data indicate that NANOG- 
    bearing mutations within the SBC motif are oncogenic and resis-tant to SPOP-mediated degradation.
    Phosphorylation of NANOG at Ser68 Blocks Its Degradation by SPOP
    Although our data identified that cancer-associated Ser68 muta-tion of NANOG abolished SPOP-mediated degradation, the mutation frequency of NANOG Ser68 is very low by our analysis from TCGA database. This triggered our exploration of other
    Figure 4. Oncogenic NANOG Mutations within SBC Are Resistant to SPOP-Mediated Degradation
    (A) Sequence alignment of NANOG and cancer associated mutation within the SPOP binding motif (SBC).
    (B) HA-SPOP and indicated FLAG-tag NANOG plasmids were co-expressed in HEK293T cells. Cell lysates were prepared for coIP and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting. (C) Binding was examined using biotinylated peptide pull-down assay and analyzed using WB.
    (D) FLAG-NANOG or S68Y were expressed in HEK293T cells. After treating cells with CHX (10 mg/mL) for indicated time intervals, protein levels of NANOG were analyzed by WB.
    (E) FLAG-NANOG or mutants were co-expressed in HEK293T cells with HA-SPOP. After treating cells with CHX (10 mg/mL) for indicated time intervals, protein levels of NANOG and SPOP were analyzed by WB.
    (F) The NANOG protein abundance in (E) was quantified by ImageJ and plotted as indicated.
    (G) FLAG-NANOG or S68Y, HA-SPOP, and His-ubiquitin were co-expressed in HEK293T cells. After treatment with MG132 (10 mM) for 6 hr, the Ni-NTA ubiq-uitination assay was performed and analyzed by WB.
    (I) Representative sphere images from each condition of DU145 cells. Scale bar, 200 mm.
    (J) Growth of DU145 cell-derived tumors in nude mice infected with lentivirus expressing indicated constructs.
    possible mechanisms in mediating the stability regulation of NANOG. A pervious study showed that NANOG could be phos-phorylated at Ser68 using high-resolution mass spectrometry (Brumbaugh et al., 2014). To investigate whether the phosphor-ylation of Ser68 affects its interaction with SPOP, we con- 
    structed a phosphorylation-mimic mutant of NANOG, S68D (Figure 5A). Our data indicated that the S68D mutant lost the abil-ity to interact with SPOP (Figure 5B). Moreover, SPOP could not promote the degradation of NANOG S68D (Figures S5A–S5C). Our data from a peptide pull-down assay showed that SPOP