Phosphorylation stabilizes Nanog by promoting its interaction with Pin1

Abstract

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body, thus holding great promise as a renewable source of cells for human therapy. The mechanisms that maintain self-renewal of ESCs remain unclear. Here we show that Nanog, a transcription factor crucial for the self-renewal of ESCs, is phosphorylated at multiple Ser/Thr-Pro motifs. This phosphorylation promotes the interaction between Nanog and the prolyl isomerase Pin1, leading to Nanog stabilization by suppressing its ubiquitination. Inhibition of Pin1 activity or disruption of Pin1-Nanog interaction in ESCs suppresses their capability to self-renew and to form teratomas in immunodeficient mice. Therefore, in addition to the stringent transcriptional regulation of Nanog, the expression level of Nanog is also modulated by posttranslational mechanisms.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Nanog interacts with Pin1 through its phosphorylated Ser/Thr-Pro motifs. (A) The endogenous Nanog is phosphorylated at Ser/Thr-Pro motifs. Endogenous Nanog was immunoprecipitated with anti-MPM2 antibody that specifically recognizes phosphorylated Ser/Thr-Pro motifs, followed by Western blotting for the levels of Nanog protein in the immunoprecipitate. (B) Mapping of the phosphorylated Ser/Thr-Pro motifs of Nanog. Cell lysates derived from mouse ESCs expressing myc-tagged WT Nanog and myc-tagged Nanog4A were immunoprecipitated with MPM2 antibody, followed by Western blotting with anti-myc antibody. (C) The interaction of the endogenous Nanog and Pin1 in mouse ESCs shown by reciprocal coimmunoprecipitation analysis. NRS and NMS refer to normal rabbit serum and normal mouse serum, respectively, that are negative controls for the specificity of antibodies used in the immunoprecipitation. IP and WB refer to immunoprecipitation and Western blotting, respectively. (D) The phosphorylated Ser/Thr-Pro motifs at the N terminus of Nanog are responsible for binding to Pin1. GST-Pin1 pull-down assay were performed using lysates of HEK 293 cells expressing myc-tagged WT Nanog, NanogS71A, Nanog3A and 2A (D), and Nanog3E (E). GST was used as negative control for the pull down assay. (E) Phosphorylation of Nanog promotes its interaction with Pin1 in ESCs. The interaction between myc-tagged Nanog4A and Pin1 is greatly reduced in ESCs as shown by coimmunoprecipitation. (F) Nanog3E (Ser52, 65, 71 to Glu) mutation disrupts the interaction between Nanog and Pin1 as shown by GST-Pin1 pull-down assay.

Fig. 2.

Identification of the phosphorylation sites of Nanog by MS. Myc-tagged Nanog expressed in 293 cells were purified and analyzed by MS. Unique peptides from Nanog were identified with a protein sequence coverage of 65%. Four phosphorylation sites of Nanog were identified at S52, S56 or S57, S65, and S77 or S78. Phosphorylations of S52 and S65 were unambiguously identified, whereas the MS/MS data do not allow us to pinpoint the exact phosphorylation sites between S56/S57 and S77/S78.

Fig. 3.

Pin1 is important for the self-renewal of mouse and human ESCs. (A) Pin1 inhibitor reduces the protein levels of Nanog but not Oct4 in mouse ESCs. The protein levels of Nanog and Oct4 in mouse ESCs were analyzed at different time points after treatment with 20 μM PiB. The time points are indicated on the top. (B) Pin1 inhibitor reduces the stability of Nanog. The protein levels of Nanog in mouse ESCs were analyzed at different time points after the treatment with cycloheximide (CHX) in the presence or absence of Pin inhibitor (PiB). (C) Pin1 inhibitor reduces the protein levels of Nanog in human ESCs. Human ESCs were mock-treated or treated with 18 μM PiB and harvested 8 and 16 h after treatment for the analysis of the Nanog and Oct4 protein levels. The time points and treatment are indicated on the top. (D) Silencing of Pin1 expression via RNAi reduces the protein levels of Nanog in mouse ESCs. Pin1, Nanog, OCT4, and tubulin are indicated. (E) Pin1 inhibitor (PiB) suppresses the self-renewal of mouse and human ESCs. Clonogenic survival assay of mouse ESCs (Left) and human ESCs (Right). ESCs were trypsinized into single cells and plated on feeder layer at low density. Twenty-four hours after plating, the cells were mock treated or treated with increasing concentrations of PiB. The number of colonies are counted at 8 d (mouse ESC) or 14 d (human ESC) after treatment. Mean value from three independent experiments are presented with error bars. (F) Knockdown of Pin1 with RNAi in mouse ESCs reduces their self-renewal potential. The number of colonies was counted 8 d after plating. Mean values from three experiments are presented with error bars.

Fig. 4.

Pin1 inhibits the ubiquitination of Nanog. (A) Proteosome-dependent pathway contributes to Nanog degradation. Mouse ESCs were mock-treated or treated with 20 μM PiB. Four hours before harvest, the PiB-treated cells were mock-treated or treated with the proteasome inhibitor ALLN. The treatments and time points are indicated on the top. The interaction between Pin1 and Nanog promotes the ubiquitination of Nanog in HEK293 cells (B) and in mouse ESCs (C). In B, HEK293 cells expressing HA-ubiquitin together with myc-tagged WT Nanog or Nanog4A were mock-treated or treated with ALLN. The ubiquitination of Nanog was revealed by immunoprecipitation with anti-HA antibody followed by immunoblotting with anti-Nanog antibody. In C, myc-tagged WT Nanog or Nanog4A was immunoprecipitated from ESCs expressing these proteins followed by immunoblotting with antiubiquitin antibody. The levels of unubiquitinated Nanog is also shown. (D) Increased Pin1 expression reduces the ubiquitination of Nanog in HEK293 cells. HEK293 cells expressing HA-ubiquitin and myc-tagged WT Nanog in the absence or presence of the overexpression of Flag-Pin1 were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-Nanog antibody. (E) Pin1 inhibitor greatly increases the ubiquitination of Nanog in mouse ESCs. Mouse ESCs expressing myc-tagged Nanog were mock-treated or treated with PiB for 6 h in the absence or presence of ALLN. The ubiquitination of Nanog was detected by immunoprecipitation with anti-myc antibody followed by immunoblotting with antiubiquitin antibody. The total amount of unubiquitinated Nanog was revealed by anti-Nanog antibody. (F) Self-renewal of ESCs expressing Nanog 4A and 3A is reduced compared with WT. ESCs expressing Nanog WT, 4A, and 3A were plated at clonal density and the colonies were stained with the alkaline phosphatase detection kit and counted (Top). Nanog4A and 3A are less stable than WT Nanog in ESCs (Bottom). ESCs expressing myc-tagged WT Nanog, Nanog4A, or Nanog3A were incubated with cycloheximide and harvested at the indicated time points. The protein levels of myc-tagged Nanog are revealed with anti-myc antibody. (G) The expression of Nanog4A triggers NanogWT to ubiquitin mediated degradation. HEK293 cells expressing HA-ubiquitin and Nanog-FLAG in the presence of NanogWT-Myc or Nanog4A-Myc, respectively, were immunoprecipitated by HA antibody and probed with an anti-FLAG antibody.

Fig. 5.

Pin1 and its interaction with Nanog is important for teratoma formation of ESCs in SCID mice. (A) Pin1 inhibitor (PiB) suppresses the teratomas formation when mixed with mouse ESCs. Mouse ESCs mixed with PiB (20 μM) were injected s.c. into right side of SCID mice. As an internal control for the potential of teratomasformation by the treated ESCs, the same number of mock-treated ESCs was implanted at the left side of the same SCID mice. Approximately 4 wk after implantation, tumors were excised and weighted. Representative image of one set of tumors derived from treated and control ESCs is shown. The ratio of the weight of the tumor derived from treated ESCs versus untreated control is shown below. Mean ratio from three independent experiments is shown with error bars. (B) Pretreatment of mouse ESCs with PiB for 24 h suppresses teratoma formation in SCID mice. Mouse ESCs were mock-treated or treated with PiB (20 μM) for 24 h before implantation. The treated ESCs were implanted on the right side of the SCID whereas the same number of control ESCs were implanted on the left side of the same mouse. The ratio of the weight of the tumor derived from treated ESCs versus untreated control is shown below. Mean ratio from three independent experiments is shown with error bars. (C) The levels of Nanog and Oct4 are dramatically reduced in the teratomas derived from PiB-treated ESCs. Protein extracts from control and PiB-treated teratomas were probed with Nanog, OCT4, and tubulin antibody. (D) The interaction between Pin1 and Nanog is important for teratomas formation by mouse ESCs. Mouse ESCs expressing Nanog4A or Nanog3A (as indicated) and control ESCs were implanted into left and right side of the same SCID mice, respectively. The ratio of the weight of the tumor-derived from treated ESCs versus untreated control is shown below. Mean values from three independent experiments are shown with error bars. (E) Pin1 inhibitor suppresses the teratomas formation of human ESCs in SCID mice. Human ESCs mixed with PiB and mock-treated control ESCs were implanted into the right and left sides of the same SCID mice. Six weeks later, the tumors were weighted and shown. The ratio of the weight of the tumor derived from treated ESCs versus untreated control is shown below.