Mechanism Of The Cellular Localization Of Sufu Regulated By Hedgehog Signaling

Hedgehog(Hh)gene was originally identified as a segment polarity gene in Drosophila.There are three Hh genes in vertebrates:Sonic HH(SHH),Indian HH(IHH),and Desert HH(DHH).Sonic HH pathway plays a critical role in the regulation in vertebrate embryonic development.Abnormal Shh pathway will lead embryo anamorphosis,birth defects and a variety of human tumors.Because it's closely linked to embryonic development and human disease,having a detailed understanding of the Shh signaling pathway will help us to regulate Shh signal better and develop clinical small molecule inhibitors of Shh signaling and reduce birth defects and human tumorigenesis.In vertebrates,Hh signal transduction is initiated through binding of the lipidated ligand to the 12-pass transmembrane protein Patched,which releases its repression on the 7-pass transmembrane protein,Smo.This allows Smo to translocate into primary cilia and activates the downstream signaling pathway.Downstream of Smo is the Gli family,which is a subset of zinc finger transcription factors,including Gli1,Gli2,and Gli3 three members.Suppressor of fused(Sufu)is one negative regulator of Hh signaling and as a partner of Gli proteins interacting with all Gli proteins in a complex.The Gli proteins are repressed by Sufu through tethering Gli in the cytoplasm,Sufu can bind to a amino-terminal region of Gli with a highly conservered SYGH motif.As Hh stimulation,Gli proteins locate into nucleus,suggesting that the ability of Sufu to anchor Gli in the cytoplasm is somehow relived.Some evidences suggest human Sufu normally shuttles between the nucleus and cytoplasm.This indicates that Sufu is able to remain bound to Gli protein in the nucleus.On the other hand,the nuclear protein SAP 18 can interact with Sufu specifically and SAP 18 is a binding partner of mSin3 protein,which in association with histone deacetylase(HDAC)forms a corepressor of transcription.So it is possible that Sufu may repress Glil mediated transcription by recruiting mSin3-HDAC corepressor in the nucleus.The mechanism of Sufu cytoplasmic-nuclear localization is not very clear.Our group previously found Sufu localizing in the nucleus depends on the Gli family of three members,acompanies GliA translocating into and GliR translocating out of the nucleus.In 18cases of human medulloblastoma tissues,Sufu level is very high and rich in the nucleus with a fifty percent.And in the normal tissues,Sufu completely localizes in the cytoplasma.Then we detected some other tumor tissues and cell lines,we also found that Sufu and Glil are in high levels in lung and breast cancers.These results confirmed our previous conclusions that Sufu is required for generating maximal signaling output of Sonic Hedgehog and acompanies GliA translocating into the nucleus.In these tumors,Sufu is in high level as is Glil,which means that Gli may have a regulation on the Sufu levels.By blocking protein synthesis with cycloheximide showed that Sufu is actually unstable in Gli null MEFs than in wt-MEFs.Therefore,the stability of Sufu has a relationship with the three members of the Gli protein family.Modifications of proteins also have an impact on its stability and localization,including phosphorylation,dephosphorylation and so on.Phosphorylation is a reversible process,in the activation of the Hh signaling pathway,Sufu will be dephosphorylated and be not stable.Sufu will more easily undergo ubiquitin-proteasomal degradation and the Hh signaling pathway will be more activated.However,the phosphatases regulating Sufu dephosphorylation have not been found.Interestingly,we found many phosphatases binding with Sufu through a proteomic approach,incliding PPP4R2.A higher protein levels binding with Sufu when is treated with Shh.Therefore,we speculate that Hh signaling promotes PPP4R2 interacts with Sufu,via changing Sufu phosphorylation state and then affecting its regulation on the Hh pathway.So we constructed a plasmid with Flag-tagged PPP4R2 gene,then we expressed epitope-tagged Sufu and PPP4R2 in HEK293T cells and performed immunoprecipitation using antibodies against the respective epitopes.We showed that Sufu immunoprecipitates contained PPP4R2.And the immunofluorescence experiments in wt-MEF cell showed that PPP4R2 and Sufu have a significant co-localization in the nucleus.Therefore,we suspect that PPP4R2 may have role in the regulation of Sufu phosphorylation and it's intracellular localization.These results mean that we found a new phosphorylase acting on Sufu and discovered a new mechanism regulating Sufu localization in the cells.We have a depper insight for Sufu as a mutual regulator of the Hh pathway.