The Mechanism Of PGC7 Regulating Activity Of PP2A Via MASTL Kinase

PGC7（also known as Dppa3 or Stella）is a small protein with 150 amino acids and no fixed structure.Therefore,we speculate that it functions mainly by interacting with other proteins.At present,the research on the function of PGC7 is mainly focused on the following aspects:PGC7 protects maternal genome and maternal imprinting from active demethylation during early embryonic development,promotes the maintenance of pluripotency,and promotes the reprogramming process of somatic cells to induced pluripotent stem cells（i PSCs）.MASTL kinase（microtubule associated serine/threonine kinase like）is a PGC7interacting protein discovered in the laboratory,which belongs to the AGC kinase family,also known as the Greatwall kinase（Gwl）.Its main function is to inhibit PP2A-B55.PP2A-B55 is a protein phosphatase 2A（PP2A）holoenzyme containing B55 family regulatory subunits.There are four subtypes of B55 regulatory subunits,which are encoded by PPP2R2A,PPP2R2B,PPP2R2C and PPP2R2D,respectively.PP2A is a widely expressed serine/threonine phosphatase,which exerts its regulatory function by dephosphorylation of key cellular molecules such as AKT,p53,c-Myc andβ-catenin.MASTL phosphorylates Arpp19and ENSA,which promotes the binding of Arpp19 and ENSA to PP2A-B55 to achieve the inhibition of PP2A-B55.MASTL-Arpp19（ENSA）-PP2A-B55 signaling pathway is highly conserved from yeast to human and has been verified in many model organisms,but whether PGC7 is involved in the regulation of this pathway has not been reported.In this study,the interaction between PGC7 and MASTL and its effect on PP2A activity were studied by immunoprecipitation（IP）,Co-immunoprecipitation（Co-IP）,nucleo-cytoplasmic separation,immunofluorescence（IF）and immunoblotting（western blot）.The results are as follows:1.The expression vectors of p3×Flag-MCS-CMV-10-MASTL and p EGFP-C1-MASTL were constructed,and the interaction between PGC7 and MASTL was verified by co-immunoprecipitation.The localization of MASTL and PGC7 was detected in NIH 3T3 mouse embryonic fibroblast cell line,and it was found that MASTL was mainly located in the nucleus,while PGC7 was distributed in both nucleus and cytoplasm,and MASTL could promote the nucleation of PGC7.Further treatment with LB-100,an inhibitor of PP2A,showed that the nucleation of PGC7 by MASTL might be caused by the inhibition of PP2A;at the same time,the localization of two subtypes of Ppp2ca/Ppp2cb of PP2A catalytic subunit was detected,and it was found that Ppp2ca/Ppp2cb was distributed uniformly in the nucleus and cytoplasm;to a certain extent,MASTL promoted the entry of Ppp2ca into the nucleus,while promotes Ppp2cb to transport out of nucleus,and PGC7 assists the effect of MASTL on the localization of Ppp2ca/Ppp2cb.2.Arpp19 and its mutants Arpp19^S62A and Arpp19^S104A were enriched by immunoprecipitation.It was found that PGC7 promoted the phosphorylation of Ser62 of Arpp19 by MASTL.At the same time,it was also found that Arpp19 was located in the cytoplasm and less in the nucleus.MASTL promoted the entry of Arpp19 into the nucleus,and this process had nothing to do with the phosphorylation of Ser62.In the further nuclear-cytoplasmic separation experiment,the relationship between the nuclear-cytoplasmic shuttle of Arpp19 and the dose of MASTL was discussed.It was found that the effect of MASTL on the entry of Arpp19 into the nucleus mainly depended on its own dose effect,rather than on the phosphorylation of Arpp19.PGC7 assists MASTL-promoted Arpp19 into the nucleus in this process through direct interaction with MASTL.3.The phosphorylated Ser 473 site of AKT is the main target site of PP2A.According to the phosphorylation level of this site,the effects of PGC7 and MASTL on the activity of PP2A were studied.It was found that PGC7 promoted the inhibition of PP2A activity by MASTL,which in turn promoted the phosphorylation of AKT.Further nuclear and cytoplasmic separation experiments showed that PGC7 in the nucleus rather than PGC7 in the cytoplasm played a major role in promoting the inhibition of PP2A activity by MASTL.4.It has been reported that MASTL is also regulated by phosphorylation of AKT.We treated the cells with MK-2206,an inhibitor of AKT,and SC79,an activator of AKT,and found that AKT activity affected the stability of MASTL and made MASTL tend to degrade easier;at the same time,we found that PGC7 significantly promoted the interaction between AKT and MASTL via several groups of Co-IP,suggesting that this PGC7 may play a role in the activation mechanism of AKT on MASTL.In conclusion,this study found that PGC7 promoted the phosphorylation of Arpp19 by MASTL and enhanced the inhibition of PP2A activity,which was beneficial to the activation of AKT.At the same time,the entry of Arpp19 into the nucleus caused by MASTL suggests that the main function of PGC7 may be to promote the inhibition of PP2A in the nucleus and the increase of AKT activity in the nucleus.Therefore,our results may provide new perspectives for elucidating the mechanism and pathway of PGC7 regulating DNA methylation.