Zinc is essential for binding of p56(lck) to CD4 and CD8alpha.
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.
Lin
R S
RS
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Rodriguez
C
C
Veillette
A
A
Lodish
H F
HF
D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism*; D016827:CD8 Antigens / Q000378:metabolism*; D019556:COS Cells; D002474:Cell-Free System; D006801:Humans; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D011485:Protein Binding; D015032:Zinc / Q000378:metabolism*
D015704:CD4 Antigens; D016827:CD8 Antigens; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015032:Zinc