Zinc is essential for binding of p56lck to CD4 and CD8α
Zinc is essential for binding of p56lck to CD4 and CD8α
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DOI：
10.1074/jbc.273.49.32878
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发表时间：
1998-12-04
影响因子：
4.8
通讯作者：
Lodish, HF
中科院分区：
文献类型：
--
作者：
Lin, RS;Rodriguez, C;Lodish, HF
蛋白酪氨酸激酶p56（lck）与T细胞共受体CD 4和CD 8 α的结合是T淋巴细胞发育和活化所必需的。p56（lck）与CD 4的结合需要CD 4的胞质结构域中的两个保守半胱氨酸残基和p56（lck）的氨基末端中的两个半胱氨酸残基，这与这四个残基配位单个金属原子的概念一致（1-5）。在这里，我们证明，Zn 2+是必不可少的复杂的形成。在体外结合反应中，Zn 2+介导p56（lck）与含有CD 4或CD 8 α胞质结构域的谷胱甘肽S-转移酶（GST）融合蛋白的结合;没有其他金属测试支持结合。用Zn 2+螯合剂1，10-O-菲咯啉或8-羟基喹啉-5-磺酸处理预先形成的GST-CD 4.p56（lck）二聚体，导致GST-CD 4从p56（lck）上解离，这与Huse等人（5）的发现一致，即Zn 2+包含在类似的复合物中。此外，我们表明，在活细胞内，CD4.p56（lck）和CD 8 α.p56（lck）的相互作用发生在锌依赖的方式。具体地，用膜可渗透的锌螯合剂预处理人Jurkat T细胞系破坏了CD4.p56（lck）复合物，用这种螯合剂处理共表达CD 8 α和p56（lck）的COS细胞同样导致CD 8 α.p56（lck）复合物解离。CD4.p56（lck）和CD 8 α.p56（lck）代表需要锌作为异源二聚化的桥的细胞内蛋白的第一个实例。
Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8 alpha 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 CD8 alpha; 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 CD8 alpha.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 CD8a and p56(lck) with such chelators likewise leads to dissociation of CD8 alpha.p56(lck) complexes. CD4.p56(lck) and CD8 alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization.