T-CELL ANTIGEN CD28 INTERACTS WITH THE LIPID KINASE PHOSPHATIDYLINOSITOL 3-KINASE BY A CYTOPLASMIC TYR(P)-MET-XAA-MET MOTIF
T-CELL ANTIGEN CD28 INTERACTS WITH THE LIPID KINASE PHOSPHATIDYLINOSITOL 3-KINASE BY A CYTOPLASMIC TYR(P)-MET-XAA-MET MOTIF
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DOI：
10.1073/pnas.91.7.2834
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发表时间：
1994-03-29
影响因子：
11.1
通讯作者：
RUDD, CE
中科院分区：
文献类型：
--
作者：
PRASAD, KVS;CAI, YC;RUDD, CE
T 细胞抗原 CD28 提供 T 细胞增殖所需的共刺激信号。没有 CD28 连接的 T 细胞受体 zeta/CD3 接合会导致无反应/无反应状态，从而暗示 CD28 控制外周抗原或肿瘤的耐受性。一个尚未解决的关键问题涉及 CD28 产生细胞内信号的机制。磷脂酰肌醇 3-激酶 (PI 3-激酶) 是一种具有 Src 同源性 2 (SH2) 结构域的脂质激酶，可与血小板衍生生长因子受体 (PDGF-R) 结合，这种相互作用对于生长因子信号传导至关重要。在本研究中，我们证明 CD28 通过其细胞质尾部内的 Y(P)MXM 基序与 PI 3 激酶结合。通过脂质激酶和HPLC分析以及杆状病毒表达的PI 3-激酶p85亚基的重构实验来检测CD28相关的PI 3-激酶。 CD28 直接与 p85 亚基结合，无需相关的 p110 亚基。使用含有 Y(P)MXM 的肽进行的定点诱变和肽竞争分析表明，PI 3-激酶与 CD28 胞质尾部内的 Y(P)MXM 基序（残基 191-194）结合。基序内 Y191 的突变导致结合完全丧失，而 M194 的突变导致结合部分丧失。结合分析表明，CD28 Y(P)MXM 基序与 p85 C 端和 N 端 SH2 结构域结合的亲和力与（在 PDGF-R 和胰岛素受体底物 1 中观察到的）相当。在信号传导方面，CD28 连接诱导 PI 3-激酶与受体的募集和结合显着增加。CD28 很可能使用 PI 3-激酶作为导致 T 细胞增殖的第二个信号，这一事件对无反应性和外周T细胞耐受。
The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that (observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.