FIGURE 6.

Lys-30 ubiquitination of SLP-76 attenuates TCR signaling. FLAG-tagged (SLP-76 (WT or K30R mutant) was transfected into J14 cells, followed by anti-CD3 stimulation for different time periods. The activation of TCR signaling molecules was determined by Western blotting. A, flow cytometry analysis of transfection efficiencies of WT SLP-76 and the K30R mutant in J14 cells. GFP was coexpressed with SLP-76 via a bicistronic construct. B, the SLP-76 K30R mutant results in enhanced Ser-376 and Tyr-145 phosphorylation of SLP-76, as well as ERK and JNK activation, upon TCR signaling. The relative phosphorylation levels were determined by densitometry analysis and are shown in C. Data are representative of three independent experiments. D, flow cytometry analysis of ERK phosphorylation in J14 cells after transfection with FLAG-SLP-76 (WT or K30R mutant). The transfected cells were stimulated with anti-CD3 antibody for 0–30 min, followed by intracellular staining of phosphorylated ERK. GFP-positive cells were gated.