Generation Of Fyb And Skap1 Double-knockout Mice And Identification Of SKAP1 Interaction Partners In T-cells

Adaptor proteins are molecules that lack transcriptional activity or intrinsic enzymatic activity,but instead,carry a variety of protein-protein interaction domains which are necessary for protein binding and facilitate the creation of larger signaling complexes.SKAP1(Src-kinase-associated phosphoprotein of 55 k Da)and FYB1(FYN binding protein)are functionally related immune adaptors that are mainly expressed in T cells and immunocytes,and FYB1-SKAP1 module acts as a key mediator in the “inside-out” signaling of T cells that mediates the activation of integrin to mediate T-cell adhesion.FYB1 constitutively associates with and stabilizes SKAP1,and knockdown of FYB1 results in a significant degradation of SKAP1 in both cell line and mouse model.While FYB1 and SKAP1 share a redundant function in integrin activation for T-cell adhesion,and the single-knockout Fyb-deficient and Skap1-deficienct mice was made and available,the double-knockout mice with deficiency in both Fyb and Skap1 would be beneficial for assessing the overall effect of FYB1-SKAP1 module in TCR-induced integrin activation,which has not yet been available.The aim of this study is to generate and breed a Fyb and Skap1 double-knockout mice model by successive crossbreeding of Fyb-deficient mice with Skap1-deficient mice.Further,to identify the binding partners of SKAP1,Co-IP(Co-immunprecipitation)of SKAP1 in Jurkat T-cells was performed,and the precipitates were subject to analysis with liquid chromatography-tandem mass spectrometry(LC-MS/MS).Results: Fyb and Skap1 double knockout mice with stable inheritance were obtained,which was confirmed at DNA level by PCR genotyping with specific primers and at protein expression level by Western-blot with specific antibodies against FYB1.The dual knockout mice do not display an immune deficient phenotype and can produce healthy double-knockout offsprings.The preliminary result of LC-MS/MS analysis with the anti-SKAP1 IPs revealed a total of 46 proteins as potential SKAP1-interacting partners in T-cells.Conclusion: In this study,Fyb and Skap1 double knockout mice with stable inheritance were obtained,which lays a foundation for the follow-up study of differences between Fyb knockout mice and Skap1/Fyb double knockout mice.The identified protein that interacts with SKAP1 protein in this study has also laid the foundation for the further study of their functions in collaboration with SKAP1 in T-cells.