SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling

Abstract

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues.

Fig. 1.

Quantitative phosphoproteomic analysis of known TCR signaling proteins. Listed is a portion of the data collected representing proteins annotated in KEGG as T-cell receptor signaling proteins. Heatmaps were calculated from five replicate experiments. The label-free heatmap represents the temporal change in the phosphorylation of proteins from wild-type SLP-76 reconstituted cells (J14–76-11) through a time course of TCR stimulation. In the label-free heatmaps, black squares represent a phosphopeptide abundance equal to the geometric mean for that phosphopeptide across all time points. Yellow represents levels of phosphorylation above the average, and blue corresponds to less than average abundance (as indicated in the color legend). Within the label-free heatmap, white dots indicate a statistically significant difference (Q value < 0.05) in the fold change in phosphopeptide abundance for that time point in the SLP-76 wild-type reconstituted cells. In the second SILAC heatmap, SILAC ratios between Y3F mutant cells (J14–2D1) and SLP-76 reconstituted cells (J14–76-11) are represented for each phosphopeptide at each time point according to the SILAC heatmap color key. Black signifies no change. Red represents reduced phosphorylation in Y3F mutant cells, and green represents elevated phosphorylation in Y3F mutant cells. White dots on SILAC heatmap squares indicate a statistically significant difference (q value < 0.05) in the comparison between Y3F mutant and SLP-76 reconstituted cell SILAC ratios for that time point. Below each heatmap time point is a separate heatmap representing the coefficient of variation (cv) for that time point. According to the cv color key, black represents 0% cv, and more orange shading represents a greater cv. Blanks in the heatmaps indicate that a clearly defined SIC peak was not observed for that phosphopeptide at that time point.

Fig. 2.

Effects of N-terminal tyrosine residues of SLP-76 on the canonical TCR signaling pathway. The canonical T-cell signaling pathway is represented with SILAC heatmap quantitation and the corresponding identified phosphorylation sites. Heatmaps were calculated from the average of five replicate experiments. White dots within a heatmap square indicate a statistically significant difference (Q value < 0.05) in the comparison between Y3F and SLP-76 reconstituted cells. SILAC heatmaps are described in detail in Fig. 1.

Fig. 3.

Quantitative phosphoproteomic analysis of known TCR signaling proteins identified in both this Y3F experiment and the previous SLP-76-deficient datasets. SLP-76 reconstituted cells (WT) were used as a control in both studies. Line plots on top of the table represent the abundance of phosphopeptides observed from J14–76-11 (SLP-76 wild-type reconstituted cells) in either the Y3F study or the SLP-76-deficient cell study. Each black line represents one phosphopeptide, and the red line represents the average peak area of all the phosphopeptides in Fig. 3 at that time point. Heatmaps for Y3F experiments were calculated from the average of five replicate experiments, and heatmaps for J14 experiments were calculated from three replicate experiments. Green represents elevated phosphorylation in Y3F mutant cells or J14 cells relative to wild-type SLP-76 reconstituted cells, and red represents decreased phosphorylation. White dots within a heatmap square indicate a statistically significant difference (Q value < 0.05) in the comparison between Y3F or SLP-76-deficient (J14) and SLP-76 reconstituted cells. The SILAC heatmaps are described in detail in Fig. 1.