Proteome Identification Of Binding-partners That Interact With Cell Polarity Protein Par3 In Jurkat Cells

Cell polarity is the fundamental characteristics of eukaryotic cells and of crucial importance to the differentiation and functions of most cells in the metazoans.The partitioning-defective 3(Par-3)is a key component in the conserved Par-3/Par-6/aPKC complex.There are mainly three isoforms of Par3,180K,150K and 100K.The evolutionarily conserved cell polarity protein Par3,a scaffold-like PDZ-containing protein,plays a critical role in the establishment and maintenance of epithelial cell polarity.Although the roles of Par3 were extensively investigated in epithelial cells,its roles in other cell types such as hematopoietic cells have scarcely been explored.Recent studies have indicated that Par-3 is a multifunctional protein, and it may be involved in other biological context in addition to establishing cell polarity.Thus we believe that the identification of binding partners of Par3 is important for understanding its role.Recently,Par3 complexes were reported being polarized in T cells.Although there are Par3 in blood cells such as Jurkat cells,the functions of Par3 in the cells remain elusive.There may be different Par3 interacting proteins in Jurkat cells.In order to obtain more information about the molecular basis of Par3 functions,we identified several potential novel binding-proteins of PDZ domains of Par3 in Jurkat cells(a T-cell line)by combining GST-pull-down approach with LC-MS/MS.The identified proteins are implicated to be involved in different cellular functions,for instance,protein metabolism,protein transportation and so on.The interaction between Par3 and these identified proteins indicates that Par3 may involve in these different biological functions.The interaction of Par3 PDZ domains with three proteins,nuclear transport protein Importin-α4,proteasome activators PA28-βand PA28-γwere further confirmed through in vitro binding assay, co-immunoprecipitation assay and immunofluorescence.Our results should not only help to uncover novel functions of this evolutionarily conserved protein Par3,but also throw new light on uncovering novel functions of the cell polarity protein Par3 in blood cells.