br A previous report showed that AMPK
A previous report showed that AMPK phosphorylates BRAF at Ser729, disrupts the heterodimerization of BRAF with KSR1, and then prevents the paradoxical activation of MEK-ERK signaling (Shen et al., 2013). We thus examined whether AMPK affects the function of NANOG by phosphorylating BRAF. To this end, we generated a phosphorylation-mimic mutant, BRAF Ser729D, and detected its interaction with NANOG. Our data showed that mutation of Ser729 to Asp (S729D) completely blocked the interaction between NANOG and BRAF (Figure 6I), suggesting that Ser729 is essential for its interaction with NANOG. Importantly, our data showed that mutation of BRAF at Ser729 lost the ability to stabilize NANOG in Lipo2000 (Figures 6J and 6K).
Functionally, the BRAF inhibitor SB590885 impaired the sphere formation and cell proliferation of DU145 cells (Figures
(C) FLAG-NANOG was co-expressed with FLAG-BRAF or K483M in HEK293T cells. After treating cells with CHX (10 mg/mL) for indicated time intervals, protein levels of NANOG and SPOP were analyzed by WB.
(D) The NANOG protein abundance in (C) was quantified by ImageJ and plotted as indicated.
(F) The NANOG protein abundance in (E) was quantified by ImageJ and plotted as indicated.
(G) DU145 cells were treated with DMSO or SB590885 (10 mM) for 4 hr, cell lysates were prepared for coIP with SPOP antibody, the associated NANOG was analyzed by WB.
(H) DU145 cells were transfected with control siRNA or BRAF siRNA. After 72 hr, cells were treated with DMSO or compound C (6.6 mM) for 4 hr. Expression levels of NANOG were analyzed by WB.
(I) FLAG-BRAFs and HA-NANOG were co-expressed in HEK293T cells. After 24 hr, cells were treated with metformin (2 mM) for 4 hr. Cell lysates were prepared for coIP and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting.
(J) FLAG-NANOG was co-expressed in HEK293T cells with indicated BRAF plasmids. After treating cells with CHX (10 mg/mL) for indicated time intervals, protein levels of NANOG were analyzed by WB.
(K) The NANOG protein abundance in (J) was quantified by ImageJ and plotted as indicated.
Figure 7. SPOP Mutation and Phosphorylation of NANOG-Ser68 Contributed to Elevated NANOG Expression in Human Prostate Tumor Specimens
(A) Representative images of NANOG immunohistochemical staining in SPOP mutated or wild-type prostate tumor specimens. scale bar (up), 50 mm; scale bar (down), 12.5 mm.
(B) Quantification of NANOG expression levels in 26 cases of SPOP wild-type and 7 cases of SPOP-mutated prostate tumor specimens. NANOG staining was scored as negative (0), weak (1), intermediate (2), or strong (3).
(C) Percentage of high NANOG expression cells in SPOP wild-type and mutated prostate tumor specimens by counting the NANOG-high cell numbers using ImageJ. **p < 0.01 (Student’s t test).
(D) Representative images of NANOG and pS68-NANOG immunohistochemical staining in SPOP wild-type prostate tumor specimens. Scale bar, 12.5 mm.
(E) Quantification of SPOP mutation and pS68-NANOG expression cases in NANOG high tumors.
(F) The correlation analysis of NANOG and pS68-NANOG protein level in prostate tumor specimens. R indicates the Spearman correlation coefficient.
(G) Model for SPOP negatively regulates NANOG.
6L, 6M, and S6J). Moreover, SB590885 led to a marked decrease in the sphere formation of DU145 cells expressing WT-NANOG, but not those expressing NANOG S68Y, indicating that BRAF largely governs PCa stem cell traits via the regulation of SPOP (Figures 6N and 6O). Taken together, these results demonstrate that BRAF directly phosphorylates NANOG at Ser68 and increases the self-renewal capacity and cell prolifera-tion of PCa cells by impairing the interaction between SPOP and NANOG.
Regulation of NANOG by SPOP in Clinical Applications We next examined whether the regulation of NANOG by SPOP is correlated to PCa clinically. To this end, we analyzed the muta-tions of the MATH domain of SPOP in 33 PCa samples by Sanger sequencing and found that 7 of the 33 cases harbored SPOP mutations (Figure S7A). Interestingly, in addition to the
well-characterized F133V, W131G, and R139K/T mutations, we identified a novel SPOP mutation, F104C, located in the MATH domain. Our data showed that the SPOP F104C mutant was also deficient in promoting NANOG degradation (Figure S7B).
The expression of NANOG in above-mentioned samples was also analyzed by immunohistochemistry (IHC). The specificity of NANOG antibody was examined by IHC assay in NANOG ectopically expressed HEK293T cells (Figure S7C). The staining of NANOG was scored as negative (0), weak (1), intermediate (2), or strong (3). We found that the protein expression of NANOG was higher in SPOP-mutated tumors than that of the SPOP-WT tumors (Figures 7A and 7B). Moreover, we found that the per-centage of cells with high expression of NANOG in SPOP mutated tissues was significantly more than that of WT tissues (Figure 7C). Surprisingly, we also found that NANOG was highly