Modulation of Lck function through multisite docking to T cell-specific adapter protein.
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.
Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Box 1105, Blindern, N-0317 Oslo, Norway. stine.granum@medisin.uio.no
Stine
S
Granum
Thorny Cesilie Bie
TC
Andersen
Morten
M
Sørlie
Marit
M
Jørgensen
Lise
L
Koll
Tone
T
Berge
Tor
T
Lea
Burkhard
B
Fleckenstein
Anne
A
Spurkland
Vibeke
V
Sundvold-Gjerstad
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D008024:Ligands; D010748:Phosphopeptides; D011948:Receptors, Antigen, T-Cell; C405691:SH2D2A protein, human; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)