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

Stine Granum,Thorny Cesilie Bie Andersen,Morten Sørlie,Marit Jørgensen,Lise Koll,Tone Berge,Tor Lea,Burkhard Fleckenstein,Anne Spurkland,Vibeke Sundvold-Gjerstad

doi:10.1074/jbc.m800871200

Abstract

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)(280) and Tyr(P)(290) phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)(505) phosphopeptide, whereas the TSAd Tyr(P)(305) peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20-30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is approximately 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.

Highlights

Activation of T cells through T cell receptor (TCR)2 triggering is tightly controlled by transient changes in protein conformation, phosphorylation status, and catalytic activities of signaling molecules
We previously reported that T cell-specific adapter protein (TSAd) is tyrosine-phosphorylated in stimulated peripheral blood mononuclear cells [12] and Jurkat T cells [17]
These data indicate that Lck is able to phosphorylate and interact via its Src homology 2 (SH2) domain with more than one of the TSAd tyrosines

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The full-length TSAd and the TSAd ⌬239 –334 cDNAs were cloned into the pEF-HA expression vector, as previously reported [14, 18]. The concentration of Lck SH2 domain in the reaction cell typically was 10 mM, and the concentrations of the phosphotyrosine peptides in the ITC syringe were 0.5 mM, all dissolved in a modified MBS buffer (50 mM MOPS, 50 mM NaCl, 2 mM dithiothreitol, pH 6.8) [36]. Kinase Assay—In vitro kinase assays were performed using various GST-TSAd constructs diluted in kinase buffer containing 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 75 mM MgCl2, 15 mM dithiothreitol, and 1–2 ␮M ATP before recombinant Lck was mixed into the samples. Recombinant GST-TSAd constructs were used at an estimated final concentration in the range of 0.175–20 ␮M, whereas concentrations between 7.5 and 200 nM full-length, active Lck have been used per kinase assay reaction

RESULTS

50 Lysate

A Jurkat T cells HA-TSAd

50 Lysate anti-HA anti-Lck

DISCUSSION