Phosphorylation of multiple CD3 zeta 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 CD3 zeta subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3 eta, is an alternatively spliced product of the same gene locus as CD3 zeta. CD3 eta lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3 zeta 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 CD3 zeta versus CD3 eta is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59fyn; p59fyn but not p56lck or p62yes is associated with each of the three TCR isoforms containing CD3 zeta 2, or CD3 eta 2, or CD3 zeta-eta. This association occurs through components of the TCR complex distinct from CD3 zeta or CD3 eta. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3 zeta residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3 zeta Tyr----Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3 zeta 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.
Koyasu
S
S
Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, Massachusetts.
McConkey
D J
DJ
Clayton
L K
LK
Abraham
S
S
Yandava
B
B
Katagiri
T
T
Moingeon
P
P
Yamamoto
T
T
Reinherz
E L
EL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017252:CD3 Complex; D002460:Cell Line; D006825:Hybridomas / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D007376:Interleukin-2; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine