Contribution of kinases and the CD45 phosphatase to the generation of tyrosine phosphorylation patterns in the T-cell receptor complex zeta chain

The zeta subunit of the T-cell receptor complex plays a crucial role in cou pling the antigen binding alpha beta and gamma delta heterodimers to the do wnstream activation pathways. Three tandem amino acid sequence motifs conta ining pairs of exactly spaced Tyr-X-X-Leu/Ile sequences, designated as Immu noreceptor Tyrosine-based Activation Motifs (ITAMs), control this function. The phosphorylated forms of ITAMs serve as docking sites for several src h omology 2 (SH2) domain containing signaling proteins. The composition of th e assembled signaling complex and the outcome of cell activation depends on the tyrosine phosphorylation pattern of the zeta polypeptide. The mechanis m that conducts the generation of various phosphorylated forms has not yet been well established. In this study we have analyzed the ability of src fa mily tyrosine kinases and the CD45 tyrosine phosphatase in determining the phosphorylation slate of the different ITAMs and the individual tyrosine re sidues of the TCR zeta chain. The intracellular part of the zeta chain was phosphorylated by src family tyrosine kinases, p56(lck) and p59(fyn) in vit ro. Synthetic oligopeptides representing full-length or half-sized ITAMs wi th a single tyrosine residue were also phosphorylated by both p56(lck) and p59(fyn). In contrast, an additional membrane proximal tyrosine residue in the human zeta chain, located outside of the ITAMs, was not phosphorylated. We also examined the activity of the CD45 phosphatase, using a panel of IT AM derivatives, in which one or both tyrosines were phosphorylated. The eff iciency of ITAM dephosphorylation by CD45 was dependent on the primary sequ ence of the oligopeptides and the position of the phosphotyrosine residues. The in vitro data suggest that the CD45 phosphatase rather than the tyrosi ne kinase(s) may control the generation of specific phosphorylation pattern s of the zeta chain during cell activation. (C) 1999 Elsevier Science B.V. All rights reserved.