| Embryonic Stem Cells(ESCs)have become the focus of research by scientists because of their characteristics,that is,they can differentiate into other different types of cells while maintaining their self-renewal ability.Mouse ESCs(mESCs)are the most studied embryonic stem cells in many species.Our previous studies have shown that the CP2 family transcription factor Tfcp2l1(Transcription factor CP2-like protein 1)is an important gene for the self-renewal of mESCs.Tfcp2l1 is a common target of LIF/STAT3,Wnt/?-catenin and FGF/MEK/ERK signaling pathwys.Up-regulation of Tfcp2l1 transcript can replace the cytokine LIF,Wnt/?-catenin signaling pathway agonist or inhibitor of FGF/MEK/ERK pathway to promote mESCs self-renewal.More importantly,compared with other targets of LIF/STAT3 signaling pathway,only down-regulation of Tfcp2l1 can impair the LIF-mediated self-renewal.In addition,overexpression of Tfcp2l1 is able to reprogram mouse Epiblast Stem Cells(mEpiSCs)back to mESCs.Therefore,Tfcp2l1 has become the core factor for maintaining and inducing pluripotency of embryonic stem cells.To date,many effectors associated with Tfcp2l1 have been identified.For example,Tfcp2l1 interacts with MTA1 protein to inhibit the emergence of differentiated cells,while stimulating the expression of Esrrb to achieve LIF-independent mESC self-renewal.However,the exact mechanism that regulates the stability of Tfcp2l1 protein is unclear.Here,we proved a new mechanism to precisely control the stability of Tfcp2l1 protein.According to the predication of the human protein reference database(http://www.hprd.org/),Tfcp2l1 may interact with ?-TrCP1(?-transducin repeat containing protein 1),which is a ubiquitin ligase.In order to verify this prediction,we treated the cells with the proteasome inhibitor MG-132.The results showed that the stability of the Tfcp2l1 protein in the treated cells was improved,suggesting that Tfcp2l1 was indeed degraded through the pathway of ubiquitination-proteasome.To further explore the regulatory effect of?-TrCP1 on Tfcp2l1,we examined the changes in the amount of Tfcp2l1 protein from two aspects: overexpression and knockdown.The results showed that up-regulation of ?-TrCP1 gene did not affect the transcription level of Tfcp2l1,but it reduced the protein level ofTfcp2l1.On the other hand,the protein level of Tfcp2l1 increased significantly after down-regulation of ?-TrCP1,but the transcription level of Tfcp2l1 was unchanged.As?-TrCP2 and ?-TrCP1 are the two key components of E3 ubiquitin ligases,we examined the effect of ?-TrCP2 on Tfcp2l1 protein.The results showed that the regulation of Tfcp2l1 protein by ?-TrCP2 was weaker than that by ?-TrCP1.The above results indicate that the stability of Tfcp211 protein is mainly controlled by ?-TrCP1.Next,we want to investigate whether there is a direct interaction between ?-TrCP1 and Tfcp2l1 from the protein level.Fist,we prepared two cell lines that over-expressed the FLAG-tagged Tfcp2l1 gene,meanwhile the HA-tagged ?-TrCP1 and ?-TrCP2 were transfected respectively.The Co-Immunoprecipitation(Co-IP)and Western blot experiments were perfoemed and showed that there is indeed a direct interaction between them.Second,we mutated the conservative binding site(DSGDNS)of ?-TrCP1 on the Tfcp2l1 protein to(DAGDNA)and then transferred them into mESCs respectively.The results of co-immunoprecipitation experiment showed that Tfcp2l1 mutant also had a weaker interaction with ?-TrCP1 than wild-type Tfcp2l1.Finally,we performed the ubiquitination experiment and found that the ubiquitination of Tfcp2l1 protein induced by ?-TrCP1 overexpression was higner in the wild-type Tfcp2l1 cells compared with Tfcp2l1 mutant cells,indicating that ?-TrCP1 degraded Tfcp2l1 protein by ubiquitination manner.Moreover,the immunofluorescence assay showed that ?-TrCP1 reduced Tfcp211 protein level mainly in the cytoplasm.The serines in the conserved binding motif of ?-TrCP1(DSGDNS)must be phosphorylated before?-TrCP1 binding.According to the results predicted by the website http://www.hprd.org/,we found that the kinase MK2(MAP kinase-activated protein kinase 2)is involved in this process.To examine whether the stability of Tfcp2l1 is controlled by MK2,we performed three experiments.First,we overexpressed HA-tagged MK2 in 46 C mESCs and found that overexpression of MK2 gene resulted in a decrease in Tfcp2l1 protein levels.Second,the MK2 inhibitor was applied.The results showed that Tfcp2l1 protein level was gradually increased.Third,we knocked down MK2 transcript and observed that down-regulation of MK2 resulted in the increasing of Tfcp2l1 protein in mESCs.Taken together,these results indicate that MK2 is a new negative regulator of Tfcp2l1 protein.Toverify whether MK2 directly regulates Tfcp2l1,we performed co-immunoprecipitation method to investigate the effect of MK2 kinase on the interaction between ?-TrCP1 and Tfcp2l1.The cell lines overexpressed Tfcp2l1 were treated with or without MK2 inhibitor,the results showd that the interaction between ?-TrCP1 and Tfcp2l1 proteins was reduced by MK2 treatment.At the same time,we found that overexpression of MK2 was able to enhance the phosphorylation level of Tfcp2l1 protein,but treatment with MK2 inhibitors reduced the phosphorylation level of Tfcp2l1.In addition,the phosphorylation level of Tfcp2l1 mutant induced by MK2 is also significantly lower than that of wild-type Tfcp2l1.These data indicate that MK2 mediates the interaction of ?-TrCP and Tfcp2l1 mainly through phosphorylation of the Tfcp2l1 protein.Since Tfcp2l1 plays an important role in maintaining the self-renewal of mouse embryonic stem cells,we finally wanted to test the effects of ?-TrCP1 and MK2 on the self-renewal-promoting function of Tfcp2l1.We designed the following experiments.First,46 C mESCs transfected with FLAG-Tfcp2l1 in combination with HA,HA-?-TrCP1,or HA-?TrCP2 were cultured in serum medium without LIF for 8 days.The alkaline phosphatase(AP)staining assay showed that the ability to sustain the number of AP positive clones in Tfcp2l1 overexpressing cells is HA> ?-TrCP2>?-TrCP1,indicating that ?-TrCP1 has a stronger negative regulatory effect on Tfcp211 than ?-TrCP2.Second,we overexpressed empty vector,wild-type Tfcp2l1 and mutant Tfcp2l1 in mESCs respectively.The results showed that the ability to maintain the number of AP positive clones was Tfcp2l1 Mutant>wild-type Tfcp2l1> empty vector control.Third,to determine the functional regulatory effect of MK2 on Tfcp2l1,we overexpressed MK2 in mESCs and found that MK2 impaired the ability of LIF to promote self-renewal.In contrast,addition of MK2 inhibitor could enhance the self-renewal ability of mESCs cultured in the low concentration of LIF.Finally,we introduced a plasmid carrying wild-type or mutant Tfcp2l1 into mESCs overexpressing the MK2 gene.After cultured for 8 days in the absence of LIF,the results showed that MK2 was able to impair the ability of wild-type Tfcp2l1,but not Tfcp21 mutant,to promote mESC self-renewal.Together,these results suggest that MK2 and ?-TrCP1 can inhibit the ability of Tfcp2l1 to maintain the undifferentiated state of mESCs.In summary,?-TrCP and MK2 negatively regulate the protein level of Tfcp2l1 and theninduce the differentiation of mESCs.Our results will expand the current understating of the regulatory network of pluripotency factors in stem cells,which will be helpful for the basic research and transformation applications of stem cells in the future. |
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