Knock-in mutation of the distal four tyrosines of linker for activation ofT cells blocks murine T cell development

The integral membrane adapter protein linker for activation of T cells (LAT ) performs a critical function in T cell antigen receptor (TCR) signal tran sduction by coupling the TCR to downstream signaling pathways. After TCR en gagement, LAT is tyrosine phosphorylated by ZAP-70 creating docking sites f or multiple src homology 2-containing effector proteins. In the Jurkat T ce ll line, the distal four tyrosines of LAT bind PLC gamma -1, Grb2, and Gads . Mutation of these four tyrosine residues to phenylalanine (4YF) blocked T CR-mediated calcium mobilization, Erk activation, and nuclear factor (NF)-A T activation. In this study, we examined whether these four tyrosine residu es were essential for T cell development by generating LAT "knock-in" mutan t mice that express the;4YF mutant protein under the control of endogenous LAT regulatory sequences. Significantly, the phenotype of 4YF knock-in mice was identical to LAT(-/-) (null) mice; thymocyte: development was arrested at the immature CB4(-)CD8(-) stage and no mature T cells were present. Kno ck-in mice expressing wild-type LAT protein, generated by a similar strateg y, displayed a normal T cell developmental profile. These results demonstra te that the distal four tyrosine residues of LAT are essential for preTCR s ignaling and T cell development in vivo.