Wilcox Heather M, Berg Leslie J
Division of Medical Sciences, Harvard University, Cambridge, Massachusetts, USA.
J Biol Chem. 2003 Sep 26;278(39):37112-21. doi: 10.1074/jbc.M304811200. Epub 2003 Jul 3.
The Tec family kinase Itk plays a critical role in signal transduction downstream of the T cell antigen receptor and has been implicated in the activation of phospholipase C-gamma1, a key regulator of calcium mobilization and extracellular signal-regulated kinase (ERK) activation. We have shown previously that Itk is regulated by an activating transphosphorylation event in which Tyr-511 in the kinase domain is phosphorylated by Lck (Heyeck, S. D., Wilcox, H. M., Bunnell, S. C., and Berg, L. J. (1997) J. Biol. Chem. 272, 25401-25408). In this study, we present evidence for another mode of regulation for Itk, the autophosphorylation of Tyr-180 in the Src homology 3 (SH3) domain. To investigate the role of Itk trans- and autophosphorylation in T cell signaling, a retroviral transduction system was used to introduce different versions of Itk into Itk-deficient primary T cells. We report that Itk mutated at either the trans- or the autophosphorylation site is unable to fully restore cytokine production and ERK activation in the Itk-deficient cells; Itk-Y511F is severely defective, whereas Itk-Y180F has partial activity. Because phosphorylation at Tyr-180 is predicted to interfere with ligand binding by the SH3 domain, an SH3 point mutant that cannot bind ligand was also examined and found to be unable to restore function to the Itk-/- cells. These data provide new insights into the complex regulation of Itk in primary T cells.
Tec家族激酶Itk在T细胞抗原受体下游的信号转导中起关键作用，并与磷脂酶C-γ1的激活有关，磷脂酶C-γ1是钙动员和细胞外信号调节激酶（ERK）激活的关键调节因子。我们之前已经表明，Itk受一种激活转磷酸化事件的调节，其中激酶结构域中的酪氨酸511（Tyr-511）被Lck磷酸化（Heyeck, S. D., Wilcox, H. M., Bunnell, S. C., and Berg, L. J. (1997) J. Biol. Chem. 272, 25401 - 25408）。在本研究中，我们提供了Itk的另一种调节模式的证据，即Src同源3（SH3）结构域中酪氨酸180（Tyr-180）的自磷酸化。为了研究Itk的转磷酸化和自磷酸化在T细胞信号传导中的作用，使用逆转录病毒转导系统将不同版本的Itk引入Itk缺陷的原代T细胞中。我们报告说，在转磷酸化或自磷酸化位点发生突变的Itk无法在Itk缺陷细胞中完全恢复细胞因子的产生和ERK激活；Itk-Y511F严重缺陷，而Itk-Y180F具有部分活性。由于预测Tyr-180处的磷酸化会干扰SH3结构域与配体的结合，因此还检查了一种不能结合配体的SH3点突变体，发现它无法恢复Itk-/-细胞的功能。这些数据为原代T细胞中Itk的复杂调节提供了新的见解。
