Alexander D R, Brown M H, Tutt A L, Crumpton M J, Shivnan E
Department of Immunology, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
Biochem J. 1992 Nov 15;288 ( Pt 1)(Pt 1):69-77. doi: 10.1042/bj2880069.
The role of cytosolic and membrane-associated phosphatases in regulating dephosphorylation of the CD3 antigen gamma-chain has been investigated using streptolysin-O-permeabilized T lymphoblasts and Jurkat T leukaemia cells. Permeabilization of T cells caused a rapid extrusion of cytosolic type 2A phosphatases, but a membrane-associated phosphorylase phosphatase activity remained inside the cells. This activity had the properties characteristic of type 2A phosphatases, being resistant to inhibition by type 1 phosphatase inhibitors, though it was inhibited in a time-dependent manner by ATP or by non-hydrolysable ATP analogues, but not by GTP, CTP, ITP or PPi. The membrane-associated type 2A phosphatase in permeabilized cells did not dephosphorylate the CD3 antigen gamma-chain, suggesting that cytosolic phosphatases dephosphorylate the gamma-chain in situ. Cross-linking the CD2 and CD3 antigens with a bivalent monoclonal antibody in the absence of cytosolic phosphatases induced marked phosphorylation of the CD3 gamma-chain, immunoprecipitated using a novel gamma-chain peptide analogue directed antiserum (TG1). Phosphorylation was inhibited by a protein kinase C (PKC) pseudosubstrate inhibitor, indicating that CD2/CD3-induced gamma-chain phosphorylation is a PKC-mediated event. Activation of T cells either with phorbol 12,13-dibutyrate or by CD2-CD3 cross-linking caused [32P]Pi incorporation into the same gamma-chain Ser residues. The site-mapping data suggested that PKC in situ may incorporate phosphate at the CD3 gamma-chain Ser-123 and Ser-126 residues, but that phosphate is rapidly lost from Ser-123 by cytosolic phosphatase action. Our findings underline the importance of the dual actions of kinases and phosphatases as potential regulators of T cell antigen-receptor complex function.
利用链球菌溶血素O通透的T淋巴母细胞和Jurkat T白血病细胞，研究了胞质和膜相关磷酸酶在调节CD3抗原γ链去磷酸化中的作用。T细胞通透化导致胞质2A型磷酸酶迅速排出，但膜相关磷酸化酶磷酸酶活性仍保留在细胞内。这种活性具有2A型磷酸酶的特征，对1型磷酸酶抑制剂的抑制具有抗性，尽管它会被ATP或不可水解的ATP类似物以时间依赖性方式抑制，但不会被GTP、CTP、ITP或PPi抑制。通透化细胞中的膜相关2A型磷酸酶不会使CD3抗原γ链去磷酸化，这表明胞质磷酸酶在原位使γ链去磷酸化。在没有胞质磷酸酶的情况下，用二价单克隆抗体交联CD2和CD3抗原会诱导CD3γ链显著磷酸化，使用一种新型γ链肽类似物定向抗血清（TG1）进行免疫沉淀。磷酸化被蛋白激酶C（PKC）假底物抑制剂抑制，表明CD2/CD3诱导的γ链磷酸化是PKC介导的事件。用佛波醇12,13 - 二丁酸酯激活T细胞或通过CD2 - CD3交联会导致[32P]Pi掺入相同的γ链丝氨酸残基。位点映射数据表明，原位PKC可能在CD3γ链的Ser - 123和Ser - 126残基处掺入磷酸，但由于胞质磷酸酶的作用，磷酸会迅速从Ser - 123丢失。我们的研究结果强调了激酶和磷酸酶的双重作用作为T细胞抗原受体复合物功能潜在调节因子的重要性。
