br Figure AMPK Promotes SPOP Mediated Destruction of NANOG
Figure 5. AMPK Promotes SPOP-Mediated Destruction of NANOG through Downregulated Ser68 Phosphorylation of NANOG
(A) Sequence alignment of phosphorylation of NANOG within the SPOP binding motif (SBC).
(B) HA-SPOP was co-expressed with FLAG-NANOG or mutations in HEK293T cells. Cell lysates were prepared for coIP and WB. Cells were treated with MG132
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bound to the peptide of 66PDSST70 in WT-NANOG but not the phosphorylated peptide of 66PDS(P)ST70 at Ser68 (Figure S5D), suggesting that the phosphorylation of NANOG at Ser68 blocked its interaction with SPOP.
To further investigate the mechanism underlying NANOG Ser68 phosphorylation, we generated an antibody that could specifically recognize the Ser68-phosphorylated NANOG (p-S68 NANOG). Our data clearly showed that the antibody specifically targeted to Ser68-phosphorylated NANOG since it did not recognize the NANOG peptide mutated at Ser68 (Figure S5E).
AMPK Attenuates the Phosphorylation of NANOG at Ser68
Next, we aimed to identify the protein kinase that involves the phosphorylation of NANOG at Ser68. Since the phosphoryla-tion of Ser68 may affect the protein stability of NANOG, it is a reasonable assumption that the kinases involved in the phosphorylation of NANOG should affect NANOG protein stability. To this end, we generated a cell line that is stably expressing NanoLuc-NANOG fusion protein and screened 244 kinase inhibitors by monitoring the NanoLuc activity. Our data showed that treatment of MG132 or MLN4924 efficiently increased the NanoLuc activity, indicating that this assay could identify the unique compound that affects NANOG stability (Figure S5F). Surprisingly, we found that the NanoLuc activity was significantly increased with the treatment of compound C (dorsomorphin), a specific inhibitor of AMPK (Figure S5F), suggesting that compound C may have an important effect on the stability of NANOG. Furthermore, the effect of com-pound C on NANOG stability was confirmed by western blotting (Figures 5C, 5D, S5G, and S5H). Importantly, we found that the regulatory role of compound C on NANOG stability is dependent on the presence of SPOP (Figures 5C and 5D). To further confirm that AMPK is involved in the regulation of NANOG stability, we expressed NANOG in AMPKa1/a2 / MEF Fer-1 and evaluated the half-life of NANOG. Our data
clearly demonstrated that the half-life of NANOG was signifi-cantly prolonged in AMPKa1/a2 / MEF cells compared to that of WT MEFs. However, compound C failed to stabilize NANOG in AMPKa1/a2 / MEF cells (Figures 5E and 5F).
We therefore examined whether the activation of AMPK could promote NANOG degradation. To this end, 2-DG, a glucose molecule that can activate AMPK by increasing the cellular con-centration of AMP/ATP was used (Wang et al., 2015). Defects in glucose metabolism create energy stress in cells and thereby activate AMPK kinase (Jones and Thompson, 2009). Our data showed that the half-life of NANOG was significantly reduced upon the treatment of 2-DG or withdrawal of glucose (Figures S5I and S5J). These data together indicate that AMPK is a nega-tive regulator of NANOG stability.
Then we examined whether AMPK affects the phosphorylation of NANOG at Ser68. Our data showed that inhibition of AMPK with compound C significantly increased the phosphorylation of NANOG at Ser68 (Figures 5G and S5K). On the other hand, AMPK activator AICAR reduced the phosphorylation of NANOG at Ser68 (Figures 5H and S5L). Moreover, inhibition of AMPK abrogated the interaction between SPOP and NANOG, while activation of AMPK strengthened the association between SPOP and NANOG (Figures 5I and S5M).
Next, we examined whether AMPK affects NANOG stability dependent on the presence of SPOP. Our data indicated that in-hibition of AMPK by compound C prolonged the half-life of NANOG in SPOP+/+, but not SPOP / DU145 cells (Figures 5C
and 5D). Consistently, AMPK affected the ubiquitination of NANOG in SPOP+/+, but not SPOP / cells (Figures 5J, 5K,
and S5N). These data indicate that AMPK affects NANOG degra-dation and ubiquitination in a SPOP-dependent manner.
Functionally, we found that inhibition of AMPK dramatically
increased the sphere-forming potential and cell proliferation in SPOP+/+ but not SPOP / DU145 cells (Figures 5L and 5M).
On the other hand, the self-renewal capacity and proliferation of DU145 cells decreased upon the activation of AMPK (Figures 5N and 5O). Meanwhile, metformin (AMPK activator) reduced the
(C) SPOP WT or KO DU145 cells were treated with compound C (6.6 mM) for 4 hr before performing the CHX (10 mg/mL) chase analysis. Expression levels of NANOG and SPOP were analyzed by WB.