Pacini S, Ulivieri C, Di Somma M M, Isacchi A, Lanfrancone L, Pelicci P G, Telford J L, Baldari C T
Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy.
J Biol Chem. 1998 Aug 7;273(32):20487-93. doi: 10.1074/jbc.273.32.20487.
The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation. Following receptor engagement, ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor (TCR). This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins. Among these is the Shc adaptor, which couples the activated TCR to Ras. Shc interaction with ZAP-70 is mediated by the Shc PTB domain. The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Phe474) of the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta. Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR-dependent gene activation. ZAP-70 could effectively phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway.
蛋白酪氨酸激酶ZAP-70在T细胞活化过程中发挥核心作用。在受体结合后，ZAP-70被招募至T细胞抗原受体（TCR）的磷酸化亚基。这一事件导致ZAP-70活化，并使其与多种信号蛋白结合。其中包括衔接蛋白Shc，它将活化的TCR与Ras偶联。Shc与ZAP-70的相互作用由Shc的PTB结构域介导。含有分离的PTB结构域的Shc突变体的抑制作用表明，Shc与ZAP-70的相互作用可能是TCR信号传导所必需的。在此，我们表明，ZAP-70上假定的Shc结合位点（跨越酪氨酸474）的点突变（Phe474）可阻止ZAP-70与Shc相互作用以及随后Shc与磷酸化ζ链的结合。该突变既不影响ZAP-70的催化活性，也不影响TCR触发诱导的蛋白质磷酸化模式。然而，Phe474 ZAP-70突变体的表达导致TCR依赖性基因活化受损。ZAP-70在体外可有效磷酸化Shc。只有包含Shc上两个Grb2结合位点的CH结构域被ZAP-70磷酸化。两个Grb2结合位点都是ZAP-70的良好底物。数据表明，ZAP-70上的酪氨酸474是TCR信号传导所必需的，并提示Shc与ZAP-70的结合以及由此导致的Shc磷酸化可能是将活化的TCR与Ras途径连接起来的一个必要步骤。
