A Study On The Interaction Between Tom1L1 And ESCRT

Endosomal sorting determines the fate of ubiquitinated proteins in endocytosis regulated by ubquitin,which participates in the uptaking of nutrient,metabolite,growth factors and hormones,maintaining the distribution characteristics of polar epithelial cell membrane domain and the recirculation of receptors with ligand.It plays an important role in supporting many physiological functions of cell,such as biological signal transduction,antigen-antibody binding reaction.ESCRT(Endosomal soreing complex required for transport)plays a key role in the mechanism of endosomal protein sorting,which is a complex associated with the sorting of ubiquitinated proteins into MVB(multelficular bodies).Tom1 L1 is involved in cell signaling and protein transporting.It is well known protein among the Toml family protain.The results showed that Tom1L1 is an important adaptor protain that regulates EGFR endocytosis.When cells are activated by EGF,Tom1L1 which phosphorylated by Src family protein kinase will binds to activated EGF receptor via Grb2 and Shc2 and will recruited to the cytoplasmic membrane.In order to understand the function of Tom1L1 protein and the mechanism of endosomal sorting,we will study the role of Tom1L1 protein in the process of endosomal sorting and the relationship between Tom1L1 protein and ESCRT complexor.These will also provide a theoretical basis for studying the disorder of proteins endosomal sorting,degradation and transportation as well as the related disease pathogenesis and the clinical treatment strategies.Method:1.Toml family proteins,Tollip,Hrs,STAM1/2 and TSG101 siRNA.The siRNAs transfected with Toml family protein,Tollip,Hrs,STAM1/2 and TSG101 were transfected into A431 cells and the lysis were analysed by western blotting.2.the relationship between Tom1L1 and ubiquitinated proteins.293T cells weretransient transfected with Myc-ubiquitin alone,or transfected with HA-Tom1,HA-Tom1L1 or HA-Tom1L2,and lysis were analysed by western blotting.3.the relationship between Tom1L1 and Hrs,STAM.A431 cells stably overexpressed HA-labeled Tom1L1 were stimulated by EGF overnight and then lysed and immunoblotted with anti-HA antibody(IP).Protein complexes were analyzed by Western blot.knock out Hrs and STAM1/2,knockout cells stimulated with EGF in the specified time respectively.Cells were analyzed by indirect immunofluorescence microscopy to detect Tyr-Tom1L1 and EGFR.4.the effect of knockout Tom1L1 on EGFR sorting.A431 cells were transfected with Tom1L1 siRNA and then treated with EGF for 30 minutes.Tom1L1 and EGFR were analyzed by indirect immunofluorescence microscopy.5.the effect of knockout Hrs on EGFR sorting.A431 cells were transfected with Hrs siRNA and then treated with EGF for 30 minutes.Cells were analyzed by indirect immunofluorescence microscopy to detect Hrs and EGFR.Result:1.The results indicate that the Toml family of proteins can recognize ubiquitinated proteins.Tom1L1 can also bind to Hrs,STAM without EGF stimulation,indicated that it is independent of Tom1L1 phosphorylation.Tom1L1 in the Hrs KO(Hrs knock out)and STAM KO(STAM 1/2 knock out)cells can be detected STAM,Hrs protein by immunoprecipitationrespectively,indicating that Tom1L1 can identify Hrs and STAM independent of the interaction of Hrs and STAM.2.Overexpression of Hrs can recruit Tom1L1 onto the endosomal body.Overexpression of Hrs can recruit Tom1L1 onto the endosomal body without Tom1L1 phosphorylated.The function of Tom1L1 were detected in mouse MEF Hrs-KOor STAM-KO cells.In these cells,the EGF stimulation can't recruit Tom1L1 from cytosal to the endosomal body,whereas in the Hrs-Resuce mouse MEF cells,EGF stimulation does.3.In the A431 cells knocked out Tom1L1,after 30 mins EGF stimulation,the size of endosomal significantly became larger,while the number of was reduced.When the cells expressed m-Tom1L1,the endosomal returned to normal.Knocking out Hrs and Tom1L1 respectively in A431,could delay the degradation of EGFR,and the effect was more obvious when knocked out Hrs and Tom1L1 simultaneously.Conclusion:1.Tom1 family of proteins can be ubiquitinated and can recognized ubiquitinated proteins.2.Overexpression of Hrs can recruit Tom1L1 onto the endosomal body,while STAM can't.Tom1L1 can't be recruited onto the endosomal body in Hrs or STAM knockout cells.This suggests that Hrs or STAM plays a decisive role in the localization process of Tom1-L1 from the cytoplasm to the endosomal.3.Knocking out Hrs or Tom1L1 respectively in A431 cells could delay the degradation of EGFR,and the effect was more obvious when knocked out Hrs and Tom1L1 simultaneously.It indicated that Tom1L1 and Hrs played an important role in the process of endosomal sorting of EGFR.