Abstract
Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8alpha is necessary for T-lymphocyte development and activation. Association of p56(lck) with CD4 requires two conserved cysteine residues in the cytosolic domain of CD4 and two in the amino terminus of p56(lck), consistent with the notion that these four residues coordinate a single metal atom (1-5). Here we demonstrate that Zn2+ is essential for complex formation. In an in vitro binding reaction, Zn2+ mediates p56(lck) association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of CD4 or CD8alpha; no other metals tested support binding. Treatment of preformed GST-CD4.p56(lck) dimers with the Zn2+ chelators 1,10-O-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid results in dissociation of GST-CD4 from p56(lck), consistent with the finding of Huse et al. (5) that Zn2+ is contained within similar complexes. Furthermore, we show that, within live cells, CD4.p56(lck) and CD8alpha.p56(lck) interactions occur in a zinc-dependent fashion. Specifically, pretreatment of the human Jurkat T-cell line with membrane permeable zinc chelators disrupts CD4.p56(lck) complexes, and treatment of COS cells co-expressing CD8alpha and p56(lck) with such chelators likewise leads to dissociation of CD8alpha.p56(lck) complexes. CD4. p56(lck) and CD8alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization.
Highlights
Binding of the protein tyrosine kinase p56lck to T-cell co-receptors CD4 and CD8␣ is necessary for T-lymphocyte development and activation
Huse et al [5] co-expressed in Escherichia coli a fusion protein consisting of maltose-binding protein fused to the CD4 cytoplasmic domain and a portion of the N terminus of p56lck; the resulting 1:1 molar complex contained one atom of zinc and formation of the complex required cysteine residues both in the CD4 cytoplasmic domain and the N terminus of p56lck
Recombinant glutathione S-transferase (GST) fused to the cytoplasmic domain of murine CD4 was produced and purified from Escherichia coli followed by immobilization on glutathione-Sepharose
Summary
Binding of the protein tyrosine kinase p56lck to T-cell co-receptors CD4 and CD8␣ is necessary for T-lymphocyte development and activation. In an in vitro binding reaction, Zn2؉ mediates p56lck association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of
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