Identification Of Pygopus-2's Interacting Proteins By Tandem Affinity Purification

Pygopus2(Pygo2) was first found in 2002 as a functional protein in Wnt signaling pathway and dowmstream partner ofβ-catenin.Pygo2 protein have two distinct conserved domains,an N-terminal homology domain(NHD) and a C-terminal PHD zinc finger motif.Pygo binds to the N-terminal domain ofβ-catenin via Lgs/BCL9 through its PHD domain.The NHD motif can activate Writ target genes independent of PHD domain.In the context of the full-length protein,the PHD of Pygo may,via its interaction to Lgs/BCL9,function to enhance target gene transcription by(1) providing a means to bring the NHD to theβ-catenin/LEF transcriptional complex and possibly target DNA to facilitate transactivation,and/or(2) anchoringβ-catenin in the nucleus thereby elevating the level of the nuclearβ-catenin/LEF complex.But the hypothesis remains to be validated.Hence,we use Tandem Affinity Purification(TAP) approaches to screen proteins that specifically interact with Pygo2.And then uncover Pygo2's functions in Wnt signaling and find more details.We constructed mammalian expression vector pNTAP-pygo2 to express Pygo2 with N-terminal CBP-SBP tag.Transfect the constructional vector to Bcap-37 cells, use G418 to isolate cells that stably express the TAP-tagged bait protein.Harvest cells, prepare protein extracts and then purify the protein complexes through two steps of affinity purification in warm conditions.The proteins interacts pygopous2 directly or indirectly can be co-purified.Apply the elution to SDS-PAGE electrophoresis and then silver stain,8 specific protein bands was detected and subject to MALDI-TOF analysis and protein database searching.The revealed candidates contains CDK8 andβ-catenin.CDK8,is known to be a component of the Mediator complex that consisting of CDK8,CyclinC,MED12,and MED13.Mediator is an evolutionarily conserved multi-protein interface between gene-specific transcription factors and the RNA polymeraseⅡgeneral transcription machinery.We identified that Pygopous2's NHD domain interacted with endogenesis CDK8 by using TAP purification.And then we constructed pCMV-3×FLAG-MED12 and pCMV-3×FLAG-MED13,co-transfected with pCMV-myc-N-pygo2 to 293T cells respectively,then subject to co-immunoprecipitation.The result showed that the interaction of N-pygo2 and MED12/MED13 is conclusively.By those studies,we conclude that the Pygo2 have double functions in Wnt signaling:recruitingβ-catenin in the nucleus and activate gene transcription.And the transcriptional activity may due to the interaction of NHD and mediator.But the hypothesis remains to be approved.In conclusion,we purified CDK8 with TAP-MS and detect that interactions of Pygo2 and mediator.This will help us to understand the complicated regulations for Pygo2 functions.At the same time,it will help us to carry out the study of Wnt signaling pathway.