Supplementary Figure 1: ZAP-70-mediated phosphorylation of LAT Y132 is a slow signal event.

a. J.CskAS Jurkat cells were treated with the PP1 analog, 3-iodo-benzyl-PP1, for various periods (sec) of time. Lysates were subjected to immunoblot analysis of p-Y171, p-Y132 of LAT or p-Y783 of PLC-γ1. Total LAT is used as a loading control. Note the same lysates were run on two separate gels to blot for p-Y171 and p-Y132. b. The relevant bands in each immunoblot were quantified by Image Lab. The signal intensity was normalized to the 0 sec time point first and then further normalized as the fraction of maximal responses. The experiments were performed seven times. The brackets represent the standard deviation (mean ± s.d; n = 7). c. Representative bar graphs of phosphorylation of Y132 by ZAP-70. Data are derived from a high-throughput phosphorylation screen using the ZAP-70 kinase domain and a peptide library spanning LAT residues 120-139, in a Y127F background, as reported in previously (Shah et al., 2016). This subset of the data from the full screen shows the impact of every amino acid substitution at residues 131 (-1 position) and 135 (+3 position) on the ability of ZAP-70 to phosphorylate Y132. Data are shown on a log₁₀-scale relative to the parent (“wild-type”) sequence (glycine at 131 and valine at 135). A positive value indicates enhancement of phosphorylation relative to the parent sequence, a value close to zero indicates no impact on phosphorylation efficiency, and a negative value indicates that the substitution reduced the efficiency of phosphorylation. The screen shows that most -1 substitutions enhance phosphorylation by ZAP-70, relative to a -1 glycine, and that ZAP-70 strongly prefers to phosphorylate substrates with a +3 hydrophobic residue. The average effect of mutations at Y132 are shown by a red dotted horizontal line to demonstrate the magnitude of the most negatively-perturbing substitutions (that is the signal floor of the assay). This high-throughput screen was done once. d. CRISPR-Cas9 was used to generate ZAP-70-deficient Jurkat cells (J.Zap70.KO) or ITK-deficient Jurkat cells (J.Itk.KO). Cells were stimulated with anti-TCR mAb (C305) at 37^oC for a time course of 1, 2, or 5 min. Lysates were then subjected to immunoblot analysis as indicated. Data are representative of four independent experiments.