Extended Data Fig. 3: The RK motif is a conserved motif in the CD3ε cytoplasmic tail.

a, The CD3ε cytoplasmic tail sequences from different vertebrate species were retrieved from UniprotKB. The following conserved motifs are highlighted: the proline-rich sequence (PRS; blue), the identified RK motif (green) and the ITAM (underlined). The RK motif is evolutionarily conserved from lobe-finned fishes to mammals. b, Schematic of the relative frequencies of the corresponding amino acids at that position among different species from a as reflected by the height of symbols. Sequence logo was generated with Weblogo Berkeley. c, Flow cytometry analysis of transduced mCD3ε and endogenous hCD3ε expression levels in H8εsh cells reconstituted by lentiviral transduction with the following murine CD3ε variants: WT, mutation in the RK motif (RKAA), double mutation in the RK motif and PRS (DOM) or a mutation with reduced capability to switch to the active TCR conformation (K76T). Murine 2B4 and human Jurkat T cells were used as controls. d, PD using Nck SH3.1-beads and cell lysates of H8εsh cells reconstituted with mCD3ε variants as indicated. Cells were either left untreated (-) or incubated for 2 h on ice with 5 μg/ml of the anti-mCD3ε antibody (+) to stabilize the TCR in its active conformation and in the presence of 10 μM PP2, to avoid phosphorylation. Lysates and bead-purified proteins were analyzed by immunoblotting. One representative experiment is shown (left panel). Relative signals (ζ/GST) on stimulated cells were normalized to its corresponding unstimulated sample (Uns) and statistical analysis was performed using unpaired Student’s t test on pooled data from six independent experiments. Mean values ± s.e.m. are indicated (right panel). *P < 0.05; **P < 0.01; NS, not significant. e, The affinity of the WT and RKAA mutant CD3ε peptides to the Lck SH3 domain was calculated by adding these unlabeled peptides stepwise to ¹⁵N labeled Lck SH3 while performing NMR.