Lu Y, Phillips C A, Bjorndahl J M, Trevillyan J M
Department of Veterans Affairs Medical Center Amarillo, TX.
Eur J Immunol. 1994 Nov;24(11):2732-9. doi: 10.1002/eji.1830241124.
The interaction of CD28 with its counter-receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen-dependent interleukin-2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross-linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28-induced, p56lck kinase-independent pathway causes tyrosine-phosphorylation of a 110-kDa substrate while recruitment of p56lck kinase activity is apparently required for CD28-induced tyrosine-phosphorylation of 97- and 68-kDa substrates as well as CD28-induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium-independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)-3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI-3 kinase. PI-3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was "activated" by cross-linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI-3 kinase with the "activated" CD28 receptor was rapid, occurring within 30 s of receptor cross-linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI-3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate-treated Jurkat B2.7 cells, but not untreated cells. Pervanadate-induced tyrosine phosphorylation of CD28 correlated with receptor association of PI-3 kinase in the absence of CD28 cross-linking, suggesting that CD28 association with PI-3 kinase uses a tyrosine phosphorylation-dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI-3 kinase in mediating the CD28 calcium-independent, cyclosporin A-insensitive costimulatory signal.
CD28与其反受体B7的相互作用可在T细胞中诱导一种共刺激信号，该信号对于防止克隆无能及促进抗原依赖性白细胞介素-2的产生是必需的。CD28共刺激信号的分子基础尚未完全明确，但涉及蛋白酪氨酸激酶（PTK）的激活。在本报告中，我们证明了Jurkat T白血病细胞上的CD28交联可导致至少两条PTK途径的激活。一条由CD28诱导的、不依赖p56lck激酶的途径可导致一种110 kDa底物的酪氨酸磷酸化，而p56lck激酶活性的募集显然是CD28诱导的97 kDa和68 kDa底物的酪氨酸磷酸化以及CD28诱导的细胞内钙增加所必需的。p110的酪氨酸磷酸化，而非p97或p68，与CD28不依赖钙的共刺激活性相关。基于其与磷脂酰肌醇（PI）-3激酶的p85调节亚基的共免疫沉淀，pp110分子被初步鉴定为PI-3激酶的催化亚基。如果在去污剂溶解之前通过完整细胞表面的交联使受体“激活”，则发现PI-3激酶蛋白和催化活性与CD28受体复合。PI-3激酶与“激活的”CD28受体的结合动力学很快，在受体交联后30秒内发生，并且至少30分钟内保持稳定。对CD28细胞质肽序列的分析揭示了一个假定的PI-3激酶src同源2结合基序和CD28酪氨酸磷酸化位点DYMNM。在过钒酸盐处理的Jurkat B2.7细胞中检测到了CD28的酪氨酸磷酸化，但未处理的细胞中未检测到。在没有CD28交联的情况下，过钒酸盐诱导的CD28酪氨酸磷酸化与PI-3激酶的受体结合相关，这表明CD28与PI-3激酶的结合使用了一种依赖酪氨酸磷酸化的机制。这些数据为CD28信号转导提供了一个模型，并支持PI-3激酶在介导CD28不依赖钙、对环孢素A不敏感的共刺激信号中发挥作用。
