Pruschy M N, Spencer D M, Kapoor T M, Miyake H, Crabtree G R, Schreiber S L
Howard Hughes Medical Institute, Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
Chem Biol. 1994 Nov;1(3):163-72. doi: 10.1016/1074-5521(94)90006-x.
The T-cell receptor (TCR) signaling pathway is initiated by regulated association of TCR chains, including the zeta chain. A recently reported method for inducing the dimerization or oligomerization of targeted proteins in cells used the TCR pathway as a test system. In cells transfected with cDNA encoding MZF3E, a chimeric receptor comprising the intracellular domain of the zeta chain and three copies of FK506-binding protein (FKBP), low concentrations of a synthetic dimer of the natural product FK506 (FK1012) activated the expression of reporter genes. We set out to examine the signaling pathway initiated by FK1012.
We characterized the effect of FK1012 on MZF3E and a second chimeric receptor, MZF1E, which contains the zeta chain and one copy of FKBP. Only MZF3E gave FK1012-activated signaling, as shown by an increase in the kinase activity associating with MZF3E, and the appearance of specific phosphotyrosine-containing proteins. Signaling required localization of MZF3E to the inner plasma membrane, and activation of gene transcription in response to FK1012 was dependent on the protein phosphatase calcineurin and the transcriptional activator NF-AT. Some signaling events in the pathway had different kinetics when activated by MZF3E instead of the TCR, however. An unexpected requirement for the prolonged activation of calcineurin was observed.
Synthetic dimerizers can be used to gain control over cellular processes that require the association of specific intracellular proteins. The TCR signaling pathway was selected as an initial test system; we show here that one can indeed activate this signaling pathway by inducing the oligomerization of the cytoplasmic tail of the zeta chain with the cell-permeable reagent FK1012.
T细胞受体（TCR）信号通路由包括ζ链在内的TCR链的调节性缔合启动。最近报道的一种在细胞中诱导靶向蛋白二聚化或寡聚化的方法将TCR通路用作测试系统。在用编码MZF3E（一种包含ζ链胞内结构域和三个FK506结合蛋白（FKBP）拷贝的嵌合受体）的cDNA转染的细胞中，低浓度的天然产物FK506的合成二聚体（FK1012）激活了报告基因的表达。我们着手研究由FK1012启动的信号通路。
我们表征了FK1012对MZF3E和第二种嵌合受体MZF1E（其包含ζ链和一个FKBP拷贝）的作用。只有MZF3E产生了FK1012激活的信号，这表现为与MZF3E相关的激酶活性增加以及含特定磷酸酪氨酸蛋白的出现。信号传导需要MZF3E定位于质膜内侧，并且响应FK1012的基因转录激活依赖于蛋白磷酸酶钙调神经磷酸酶和转录激活因子NF-AT。然而，当由MZF3E而非TCR激活时，该通路中的一些信号事件具有不同的动力学。观察到对钙调神经磷酸酶的延长激活有意外的需求。
合成二聚体可用于控制需要特定细胞内蛋白缔合的细胞过程。TCR信号通路被选作初始测试系统；我们在此表明，确实可以通过用细胞可渗透试剂FK1012诱导ζ链胞质尾的寡聚化来激活该信号通路。
