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  • br D The NANOG protein abundance in

    2020-08-12


    (D) The NANOG protein abundance in (C) was quantified by ImageJ and plotted as indicated.
    (E) AMPK WT and KO MEF 66-81-9 were infected with lentivirus expressed NANOG, then treated with compound C (6.6 mM) for 4 hr before performing the CHX
    (F) The NANOG protein abundance in (E) was quantified by ImageJ and plotted as indicated.
    (G) DU145 cells were treated with compound C (6.6 mM) for 4 hr; cell lysates were prepared for coIP with NANOG antibody and WB.
    (H) DU145 cells were treated with AICAR (2 mM) for 4 hr; cell lysates were prepared for coIP with NANOG antibody and WB.
    (I) DU145 cells were treated with DMSO or compound C (6.6 mM) for 4 hr; cell lysates were prepared for coIP with SPOP antibody, and the associated NANOG was analyzed by WB.
    (J and K) SPOP WT or KO DU145 cells were infected with lentivirus expressed His-Ub before being treated with compound C (6.6 mM) or 2-DG (25 mM) for 4 hr. cell lysates were IP by NANOG antibody and the ubiquitinated NANOG was analyzed by WB.
    (L) Representative sphere images from each condition of DU145 cells. The SPOP WT or KO DU145 cells were maintained in DMEM supplemented with 5 mM glucose and treated with DMSO or compound C (3.3 mM). Scale bar, 200 mm.
    (P) Representative sphere images from each condition of DU145 cells. The DU145 cells were treated with metformin (2 mM). Scale bar, 100 mm.
    M
    Figure 6. Phosphorylation of NANOG at Ser68 within SBC Motif by BRAF Blocks SPOP-Mediated Destruction of NANOG
    (A) FLAG-NANOG was co-expressed with the indicated kinases in HEK293T cells. The protein level of pS68-NANOG was analyzed by WB.
    (B) FLAG-NANOG or S68A was co-expressed with BRAF in HEK293T cells. The protein level of pS68-NANOG was analyzed by WB.
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    sphere formation of the DU145 cells expressing WT-NANOG, but not those overexpressing cancer-related NANOG S68Y mutant (Figures 5P and 5Q), which established the concept that AMPK regulates PCa stem cell traits in a SPOP-dependent manner. These results together suggest that AMPK affects the stemness of PCa cells via regulating NANOG phosphorylation at Ser68.
    Phosphorylation of NANOG at Ser68 by BRAF
    As AMPK activation reduced the phosphorylation of NANOG at Ser68, it is unlikely that AMPK is the protein kinase that directly phosphorylates NANOG. To identify the protein kinase that is directly involved in the phosphorylation of NANOG at Ser68, we searched the online website (www.phosphonet.ca) and found that multiple kinases were potential candidates, including casein kinase, Raf Kinase, GSK-3b, and AKT. To determine which kinase is the right one, we co-expressed NANOG with these kinases, respectively, and the cells were treated with MG132 to avoid the influence from protein degradation. The phosphorylation of NANOG was examined using p-S68 anti-body. Our data showed that BRAF kinase, but not other kinases listed above, specially increased the phosphorylation of NANOG at Ser68 (Figures 6A and 6B). In parallel, treatment with BRAF kinase inhibitor AZ628 or LY03009120 inhibited the phosphorylation of NANOG Ser68 (Figure S6A). Kinase dead mutant K483M (BRAF) failed to promote the phosphorylation of NANOG at Ser68 (Figure S6B), suggesting that the kinase activity of BRAF is required for NANOG phosphorylation. Furthermore, we found that BRAF can bind to NANOG in cells (Figure S6C). Together, these data suggest that BRAF is a pro-tein kinase that accounts for the phosphorylation of NANOG at Ser68.
    Next, we examined whether BRAF affects the protein stability of NANOG. Our data showed that the expression of BRAF, but not kinase dead mutant K483M, strongly increased the protein level and prolonged the half-life of NANOG (Figures 5D and 6C). On the other hand, knockdown of BRAF reduced the NANOG protein level (Figure S6D). The half-life of NANOG was
    also shortened with the treatment of BRAF inhibitor AZ628 and SB590885 (Figures S6F and S6G).
    Since BRAF can phosphorylate NANOG at Ser68, we exam-ined whether BRAF affects the interaction between NANOG and SPOP. Our data showed that inhibition of BRAF increased the interaction between SPOP and NANOG (Figure 6G) and thereby promoted NANOG ubiquitination in DU145 cells (Fig-ure S6E). Consistently, the BRAF inhibitor had a minor effect on the half-life of NANOG in the SPOP / DU145 cells (Figures 6E and 6F), suggesting that BRAF affects NANOG stability in a SPOP-dependent manner.
    Furthermore, in vitro phosphorylation assay demonstrated that BRAF could phosphorylate NANOG at Ser68 and the phos-phorylation of NANOG at Ser68 prevented the interaction be-tween SPOP and NANOG in vitro (Figures S6H and S6I).
    Phosphorylation of BRAF at Ser729 by AMPK Blocks Its Interaction with NANOG
    We next examined whether the effect of AMPK on NANOG phos-phorylation is dependent on BRAF. Our data showed that knock-down of BRAF abolished the inhibitory effect on the protein level of NANOG exerted by AMPK signaling (Figure 6H), suggesting that AMPK may affect the NANOG stability via BRAF.