Specific Interaction Between IFT88 And DGK? Is Required For Hedgehog Signaling

Hedgehog signaling pathway plays a major role of regulation and control in the procedure of organization developing and cell differentiation,in the mean time,it has intimate relationship with embryo development,organ regeneration and cancer.In the early 1990 s,many members of Hedgehog signaling pathway have been regarded as the key factors for regulating embryo development.Research manifested that blocking or abnormal activating of the Hedgehog signaling pathway may induce the geneogenous developing malformation and tumorigenesis.Therefore,going deep research on Hedgehog signaling pathway will benefit the understanding of the relationship between signaling pathway with embryo or with tumor and then provide basis to clinical therapy.The Hedgehog signal pathway is highly conservative in evolution.It mainly consists of the secretive glycoprotein ligand Hedgehog,the twelve-transmembrane receptor Ptched(Ptch),the seven-transmembrane receptor Smoothened(Smo),downstream transcription factor Gli(inculing three transcription factor Gli1,Gli2 and Gli3)and other downsteam target gene.Research demonstrated that when Hedgehog ligand gene does not exist,Ptch1 will suppress the activity of Smo and the protein coded by Gli,Gli2 and Gli3 will form as receptor by proteosome.This process will supress the activity of Hedgehog signaling pathway.When Hedgehog existed,it will bind to Ptch1,releasing the repression of Ptch1 to Smo,then Smo will transmit the signal to downtream protein Gli,blocking the forming process of proteosome and finally Gli could activate as full-length activator the expresson of downstream target protein Gli1 and Ptch1 of Hedgehog signaling pathway.In addition,primary cilia is the key position for Hedgehog signal transduction of vertebrates while it depends on intraflagellar transport(IFT)to generate and maintain.IFT divides into anterograde IFT,which is from the basal body to the tip of primary cilia and retrograde IFT,which is from the tip to the basal body of primary cilia.Both these two IFT are paticipated by IFT protein.The two-way transportation is required a special transport unit which is composed by IFT motor protein Kinesin-? and Dynein and IFT complex A and B.Regularly expressed Kinesin-2,Dynein and the IFT protein are significant for the formation and maintenance of the primary cilia and the Hedgehog signaling pathway.However,how the IFT regulates the Hedgehog signaling pathway? Is there any unknown protein which is responsible for this regulation? These questions remain unknown.According to our preliminary research,we aim to find the binding protein of IFT through immunoprecipitation(IP)and mass spectrometry.We found one of the IFT B complex,IFT88 could bind to DGK ?,which is a family member of DGK.In order to learn the binding mechanism of IFT88 and DGK,we expressed the IFT88 protein and seven member of DGK family(DGK??DGK??DGK??DGK??DGK??DGK??DGK?)in 293 T cell line and do co-IP experiment.The results show that IFT88 can bind to DGK? and other six DGK members specifically.Therefore,we want to know whether this interaction will effect the Hedgehog signaling pathway through primary cilia.In order to check that,we designed the siRNA which can knockdown DGK? and detected the protein level of Gli1 inner the cell when the Hedgehog signaling pathway was activated.We find that the knockdown of DGK? can obviously reduce the mRNA level and the protein level of Gli1.This indicated that the activation of Hedgehog singaling pathway was repressed so we deduced that DGK?could upregulate Hedgehog signaling pathway.To confirm this conjecture,we use ShhN ligand and activitor of Smo,PURM to incubate with WT-MEFS cell seperately.Through real-time PCR,we draw a conclusion that the effect of these two method to the activity of Hedgehog signaling pathway is different: when Hedgehog signaling pathway is activated by ShhN ligand,the two DGK inhibitors can both reduce the activity of the Hedgehog signaling pathway.However,when Hedgehog signaling pathway is activated by the Smo agonist PURM,neither of the two DGK inhibitors can reduce the activity of the Hedgehog signaling pathway.This result suggests that DGK protein could regulate Hedgehog signaling pathway between Ptch1 and Smo.In order to do the functional study of DGK? in primary cilia,we compared the MEF cell of DGK? knockout mouse and the wild type by immunofluorescence.We observed these two MEF cells by confocal microscope,mainly focused on the development of primary cilia and the expression quantity of IFT88 in primary cilia.The result showed significant decrease of cell basal zone of DGK? knockout MEF cells when compared with the WT MEF cell,meanwhile,the expression quantity of ift88 in primary cilia also decreased significantly.According to the above results,we further deduced that DGK knockdown could suppress Hedgehog signaling pathway.This function may have relationship with the development of primary cilia and abnormal IFT.This may provide new evidence for the hypothesis that DGK could regulate the activation of Hedgehog signaling pathway.