Siegel J N, June C H, Yamada H, Rapp U R, Samelson L E
Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD 20814.
J Immunol. 1993 Oct 15;151(8):4116-27.
The c-Raf-1 serine/threonine kinase is an important component of signal transduction pathways mediating the effects of a variety of growth factors. In activated T cells, IL-2 has been shown to induce activation of c-Raf-1, but c-Raf-1 has not previously been shown to be activated through the T-cell receptor (TCR) in resting G0 T cells. Using a sensitive immune complex kinase reaction, we show that cross-linking of the stimulatory and costimulatory receptors CD3, CD4, or CD28 induces c-Raf-1 activation in highly purified resting peripheral blood human T cells. In contrast, cross-linking the nonstimulatory receptor CD45 did not induce c-Raf-1. Surprisingly, although earlier studies had shown delayed kinetics in response to Thy-1 stimulation in murine cells, c-Raf-1 activation in response to CD3 cross-linking was one of the earliest measurable events. In spite of its early kinetics, c-Raf-1 activation was found to be downstream of several other early signal transduction events, including activation of a tyrosine kinase and a tyrosine phosphatase. Several lines of evidence suggest that activation of c-Raf-1 in response to TCR stimulation may be PKC-dependent: first, phorbol esters are extremely potent activators of c-Raf-1 in human T cells; second, the kinetics of accumulation of products of phosphatidylinositol hydrolysis coincides with the kinetics of c-Raf-1 activation; and third, physiologic activation of the PLC/PKC pathway through a transfected, G-protein-coupled receptor HM1 induced similar levels of c-Raf-1 activation with a similar time course. We conclude that c-Raf-1 activation is tightly coupled to TCR stimulation and may participate in signal transduction pathways in resting, G0 T cells. The observation that the HM1 receptor can also activate c-Raf-1 suggests that T cells have the capability to utilize both tyrosine kinase-dependent and tyrosine kinase-independent mechanisms of c-Raf-1 activation.
c-Raf-1丝氨酸/苏氨酸激酶是介导多种生长因子作用的信号转导通路的重要组成部分。在活化的T细胞中，白细胞介素-2已被证明可诱导c-Raf-1的活化，但此前尚未证明c-Raf-1可通过静息G0期T细胞中的T细胞受体（TCR）被激活。使用灵敏的免疫复合物激酶反应，我们发现刺激和共刺激受体CD3、CD4或CD28的交联可在高度纯化的静息外周血人T细胞中诱导c-Raf-1的活化。相比之下，非刺激受体CD45的交联并未诱导c-Raf-1的活化。令人惊讶的是，尽管早期研究表明小鼠细胞对Thy-1刺激的反应动力学延迟，但对CD3交联的反应中c-Raf-1的活化是最早可测量的事件之一。尽管其动力学较早，但发现c-Raf-1的活化位于其他几个早期信号转导事件的下游，包括酪氨酸激酶和酪氨酸磷酸酶的活化。几条证据表明，TCR刺激后c-Raf-1的活化可能依赖蛋白激酶C（PKC）：首先，佛波酯是人类T细胞中c-Raf-1的极强激活剂；其次，磷脂酰肌醇水解产物的积累动力学与c-Raf-1活化的动力学一致；第三，通过转染的G蛋白偶联受体HM1对PLC/PKC途径的生理激活在相似的时间进程中诱导了相似水平的c-Raf-1活化。我们得出结论，c-Raf-1的活化与TCR刺激紧密相关，可能参与静息G0期T细胞中的信号转导通路。HM1受体也可激活c-Raf-1这一观察结果表明，T细胞有能力利用酪氨酸激酶依赖性和酪氨酸激酶非依赖性的c-Raf-1活化机制。
