The C terminus of T cell‐specific adapter protein (TSAd) is necessary for TSAd‐mediated inhibition of Lck activity

Abstract

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, is expressed in activated T cells. The function of TSAd is as yet unknown. We previously showed that TSAd may modulate T cell receptor-triggered signaling events. TSAd contains a Src homology (SH)2 domain, ten tyrosines and a C-terminal proline-rich region. Here, we show that human TSAd interacts with Lck through the Lck SH2 and SH3 domains and is a substrate for Lck. The TSAd C terminus, including the proline-rich region and five tyrosines, is both necessary and sufficient for TSAd interaction with and phosphorylation by Lck. Expression of TSAd in Jurkat TAg cells results in hyperphosphorylation of endogenous Lck on Y394 and to an even larger extent on Y505, resulting in a reduced Y394/Y505 phosphorylation ratio in these cells. Furthermore, full-length TSAd, but not TSAd lacking the C terminus, inhibits the hyperactive Lck Y505F mutant when both are expressed in Jurkat T cells. In contrast, expression of the TSAd C terminus alone is sufficient to inhibit Lck Y505F in phosphorylating its substrates in Jurkat T cells. Our results indicate that the TSAd C terminus is essential for inhibition of Lck activity by TSAd, and suggest a mechanism for how TSAd may inhibit early T cell activation events.

Modulation of Lck Function through Multisite Docking to T Cell-specific Adapter Protein

“…We previously reported that phosphorylation of both Lck Tyr 394 and Tyr 505 is increased in Jurkat T cells stably expressing TSAd (17). This is in accordance with Marti et al (24), who showed that intact TSAd promotes phosphorylation of Lck Tyr 394 in primary mouse CD4 ϩ T cells.…”

“…cDNA with encoding TSAd deleted for aa 239 -256 was generated using megaprimer PCR. The pGEX-6P1 Lck SH3 domain construct was previously described (17). The pEF-Lck, pEF-Lck R154K, and pGEX-3T Lck SH2 constructs were generous gifts from Dr. Tomas Mustelin.…”

“…The following polyclonal antibodies were used: anti-LAT and anti-Tyr(P) 319 Zap-70 (Santa Cruz Biotechnology), anti-Tyr(P) 394 -Lck (anti-Tyr 416 -Src family) (Cell Signaling Technology, Beverly, MA), anti-GST (GE Healthcare), and normal rabbit serum and anti-TSAd antibodies raised against synthetic peptides of TSAd (12,17) or the recombinant C terminus of TSAd (15). As secondary antibodies, we used horse peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).…”

“…Lack of TSAd expression in mice leads to hypoproliferation of T cells (19) and development of systemic autoimmune disease (25). When overexpressed in Jurkat T cells, TSAd inhibits proximal signal transduction events by modulating Lck activity (17,18).…”

“…We previously reported that TSAd is tyrosine-phosphorylated in stimulated peripheral blood mononuclear cells (12) and Jurkat T cells (17). Co-expression with Lck is required for phosphorylation of TSAd upon tyrosine in 293T cells (17,18) and To determine which of the TSAd tyrosines could be phosphorylated, we made single Tyr to Phe TSAd mutants for all 10 tyrosines and expressed them in 293T cells together with Lck. Immunoprecipitates (IPs) of TSAd from these cells were examined for phosphorylation by immunoblotting using anti-Tyr(P) antibody.…”