Association of p59(fyn) with the T lymphocyte costimulatory receptor CD2

Binding of the Fyn Src homology (SH) 3 domain is regulated by the Fyn SH2 domain

Huamao Lin, Jill E. Hutchcroft, Christopher E. Andoniou, Malek Kamoun, Hamid Band, Barbara E. Bierer

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Human CD2 is a 50-55-kDa cell surface receptor specifically expressed on the surface of T lymphocytes and NK cells. Stimulation of human peripheral blood T cells with mitogenic pairs of anti-CD2 monoclonal antibodies (mAbs) is sufficient to induce interleukin-2 production and T cell proliferation in the absence of an antigen-specific signal through the T cell receptor. CD2 has been shown previously to associate physically with the Src family protein-tyrosine kinases p56(lck) and p59(fyn). We now report that stimulation of T cells with mitogenic pairs of anti-CD2 mAbs enhanced the association of the Fyn polypeptide with the CD2 complex, whereas stimulation with single anti-CD2 mAb had minimal effect. Using glutathione S-transferase (GST) fusion proteins, we found that CD2 bound to the Src homology (SH) 3 domain of Fyn. Interestingly, the CD2-Fyn association was negatively regulated by the Fyn SH2 domain; CD2 bound poorly to GST fusion proteins expressing both the SH2 and SH3 domains of Fyn. However, the inhibitory effect of the Fyn SH2 domain on binding of the Fyn SH3 domain to CD2 was relieved by peptides containing a phosphorylated YEEI sequence that bound directly to the Fyn SH2 domain. In addition, we found that the ability of the Fyn SH2 domain to precipitate tyrosine-phosphorylated proteins, including the CD3ζ chain, was enhanced after T cell stimulation with mitogenic pairs of CD2 mAbs. Finally, overexpression of a mutated Fyn molecule, in which the ability of the Fyn SH2 domain to bind phosphotyrosine-containing proteins was abrogated, inhibited CD2-induced transcriptional activation of the nuclear factor of activated T cells (N-FAT), suggesting a functional involvement of the Fyn SH2 domain in CD2-induced T cell signaling. We thus propose that stimulation through the CD2 receptor leads to the tyrosine phosphorylation of intracellular proteins, including CD3ζ itself, which in turn bind to the Fyn-SH2 domain, allowing the direct association of the Fyn SH3 domain with CD2 and the initiation of downstream signaling events.

Original languageEnglish (US)
Pages (from-to)19914-19921
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number31
DOIs
StatePublished - Jul 31 1998
Externally publishedYes

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Costimulatory and Inhibitory T-Cell Receptors
src Homology Domains
T-cells
Monoclonal Antibodies
Association reactions
Proteins
Glutathione Transferase
T-Lymphocytes
Tyrosine
Fusion reactions
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
NFATC Transcription Factors
Cell signaling
Peptides
Phosphotyrosine
Phosphorylation
src-Family Kinases
Cell proliferation
Cell Surface Receptors
T-Cell Antigen Receptor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Association of p59(fyn) with the T lymphocyte costimulatory receptor CD2 : Binding of the Fyn Src homology (SH) 3 domain is regulated by the Fyn SH2 domain. / Lin, Huamao; Hutchcroft, Jill E.; Andoniou, Christopher E.; Kamoun, Malek; Band, Hamid; Bierer, Barbara E.

In: Journal of Biological Chemistry, Vol. 273, No. 31, 31.07.1998, p. 19914-19921.

Research output: Contribution to journalArticle

Lin, Huamao ; Hutchcroft, Jill E. ; Andoniou, Christopher E. ; Kamoun, Malek ; Band, Hamid ; Bierer, Barbara E. / Association of p59(fyn) with the T lymphocyte costimulatory receptor CD2 : Binding of the Fyn Src homology (SH) 3 domain is regulated by the Fyn SH2 domain. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 31. pp. 19914-19921.
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abstract = "Human CD2 is a 50-55-kDa cell surface receptor specifically expressed on the surface of T lymphocytes and NK cells. Stimulation of human peripheral blood T cells with mitogenic pairs of anti-CD2 monoclonal antibodies (mAbs) is sufficient to induce interleukin-2 production and T cell proliferation in the absence of an antigen-specific signal through the T cell receptor. CD2 has been shown previously to associate physically with the Src family protein-tyrosine kinases p56(lck) and p59(fyn). We now report that stimulation of T cells with mitogenic pairs of anti-CD2 mAbs enhanced the association of the Fyn polypeptide with the CD2 complex, whereas stimulation with single anti-CD2 mAb had minimal effect. Using glutathione S-transferase (GST) fusion proteins, we found that CD2 bound to the Src homology (SH) 3 domain of Fyn. Interestingly, the CD2-Fyn association was negatively regulated by the Fyn SH2 domain; CD2 bound poorly to GST fusion proteins expressing both the SH2 and SH3 domains of Fyn. However, the inhibitory effect of the Fyn SH2 domain on binding of the Fyn SH3 domain to CD2 was relieved by peptides containing a phosphorylated YEEI sequence that bound directly to the Fyn SH2 domain. In addition, we found that the ability of the Fyn SH2 domain to precipitate tyrosine-phosphorylated proteins, including the CD3ζ chain, was enhanced after T cell stimulation with mitogenic pairs of CD2 mAbs. Finally, overexpression of a mutated Fyn molecule, in which the ability of the Fyn SH2 domain to bind phosphotyrosine-containing proteins was abrogated, inhibited CD2-induced transcriptional activation of the nuclear factor of activated T cells (N-FAT), suggesting a functional involvement of the Fyn SH2 domain in CD2-induced T cell signaling. We thus propose that stimulation through the CD2 receptor leads to the tyrosine phosphorylation of intracellular proteins, including CD3ζ itself, which in turn bind to the Fyn-SH2 domain, allowing the direct association of the Fyn SH3 domain with CD2 and the initiation of downstream signaling events.",
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AU - Hutchcroft, Jill E.

AU - Andoniou, Christopher E.

AU - Kamoun, Malek

AU - Band, Hamid

AU - Bierer, Barbara E.

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