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

S. Koyasu, David McConkey, L. K. Clayton, S. Abraham, B. Yandava, T. Katagiri, P. Moingeon, T. Yamamoto, E. L. Reinherz

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3375-3381
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number5
StatePublished - 1992
Externally publishedYes

Fingerprint

Phosphorylation
T-Cell Antigen Receptor
Tyrosine
Proto-Oncogene Proteins c-fyn
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
Association reactions
CD3 Antigens
Signal transduction
Mutagenesis
T-cells
Receptor Protein-Tyrosine Kinases
Lymphocyte Activation
Site-Directed Mutagenesis
In Vitro Techniques
Electrophoresis
Sodium Dodecyl Sulfate
Protein-Tyrosine Kinases
Interleukin-2
Polyacrylamide Gel Electrophoresis
Signal Transduction

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Phosphorylation of multiple CD3ζ tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation. / Koyasu, S.; McConkey, David; Clayton, L. K.; Abraham, S.; Yandava, B.; Katagiri, T.; Moingeon, P.; Yamamoto, T.; Reinherz, E. L.

In: Journal of Biological Chemistry, Vol. 267, No. 5, 1992, p. 3375-3381.

Research output: Contribution to journalArticle

Koyasu, S, McConkey, D, Clayton, LK, Abraham, S, Yandava, B, Katagiri, T, Moingeon, P, Yamamoto, T & Reinherz, EL 1992, 'Phosphorylation of multiple CD3ζ tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation', Journal of Biological Chemistry, vol. 267, no. 5, pp. 3375-3381.
Koyasu, S. ; McConkey, David ; Clayton, L. K. ; Abraham, S. ; Yandava, B. ; Katagiri, T. ; Moingeon, P. ; Yamamoto, T. ; Reinherz, E. L. / Phosphorylation of multiple CD3ζ tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 5. pp. 3375-3381.
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AU - Koyasu, S.

AU - McConkey, David

AU - Clayton, L. K.

AU - Abraham, S.

AU - Yandava, B.

AU - Katagiri, T.

AU - Moingeon, P.

AU - Yamamoto, T.

AU - Reinherz, E. L.

PY - 1992

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N2 - 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.

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