Fig. 8: Monophosphorylated CD3δ recruits SHP-1.
a, Peptide sequences corresponding to the cytoplasmic tails of mouse CD3δ and CD3γ. Red amino acids are not conserved between CD3δ and CD3γ; ITAMs are marked in gray, phosphates are indicated with a circled ‘p’ and N-terminal biotin with a ‘bio’. b,c, SILAC ratio and the number of identified peptides for SHP-1 (b) and ZAP70 (c) on quantification by MS–MS. T cell lysates were incubated with doubly phosphorylated, unphosphorylated or singly phosphorylated peptides. The SILAC ratio indicates the relative amounts of a protein bound to one peptide in comparison to the amounts of the same protein bound to the other peptide. d, The experiments in b and c were repeated, purified proteins were separated by SDS–PAGE and visualized using immunoblotting (n = 2). e, Schematic representation of the CARs with a mutation in the N-terminal tyrosine leaving functional just the C-terminal tyrosine. f,g, Jurkat (n = 3) (f) and primary human T cells (n = 3 for UTD, n = 5 for BBδ dimer, n = 6 for BBδFY dimer and n = 4 for BBδFY monomer) (g). Each dot represents a healthy donor transduced with the indicated CARs. Cells were stimulated with pervanadate for 5 min to achieve maximum phosphorylation and the CARs were immunoprecipitated. Purified proteins were separated by SDS–PAGE and visualized using immunoblotting. The ratio of SHP-1 and CAR was calculated. Data are represented as mean ± s.d. One-way ANOVA followed by Dunnett’s multiple-comparison test was used.
