Phosphorylation of multiple CD3ζ tyrosine residues leads to formation of pp21 in vitro and in vivo: Structural changes upon T cell receptor stimulation

T lymphocyte activation resulting from antigen recognition involves a protein tyrosine kinase pathway which triggers phosphorylation of several cellular substrates including the CD3ζ subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3η, is an alternatively spliced product of the same gene locus as CD3ζ. CD3η lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3ζ and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phosphorylation studies herein demonstrates that Tyr-132 is required for the formation of pp21. Moreover, the differential phosphorylation of CD3ζ versus CD3η is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59^fyn; p59^fyn but not p56^lck or p62^yes is associated with each of the three TCR isoforms containing CD3ζ₂, CD3η₂, or CD3ζ-η. This association occurs through components of the TCR complex distinct from CD3ζ or CD3η. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3ζ residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3ζ Tyr → Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3ζ upon TCR stimulation as reflected by alteration in its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis may affect subsequent events such as receptor desensitization, receptor movement, and/or protein associations.