Regulation of the p38 MAPK signaling pathway in Th1 development and Th1-type inflammatory diseases

辅助性T细胞1 （Th1）的异常活化在过敏反应和自身免疫性疾病中起致病作用。介导Th1细胞功能和自身免疫的关键细胞因子是IFNγ。我们之前已经克隆了人类应激响应MAPKKK、MTK1及其激活因子GADD45B和γ，并表明这些分子对于Th1细胞的效应功能是不可或缺的：在Th1细胞分化过程中，GADD45β/γ的表达被诱导，进而通过MTK1激活p38 MAPK信号通路，促进Th1细胞中IFNγ的产生。本研究的目的是：(1)阐明gadd45诱导的MTK1激活的调控机制；(2)开发抑制MTK1-p38信号传导的新方法。1)我们剖析了GADD45蛋白激活MTK1的分子机制。MTK1 N端与其c端片段结合，从而抑制c端激酶结构域。这种N-C相互作用被GADD45与MTK1 n端GAD的结合所破坏。GADD45结合还通过包含一个螺旋结构域诱导MTK1二聚化，这是MTK1在激酶激活环中Thr-1493处的反式自磷酸化所必需的。因此，我们得出结论，GADD45结合诱导MTK1 N-C在Thr-1493处解离、二聚化和自磷酸化，导致激酶催化结构域的激活。2)在哺乳动物MAPKKs的三个主要亚家族（MEK1/2、MKK4/7和MKK3/6）中发现了一个保守的对接位点，称为DVD。DVD位点与它们特定的上游MAPKKKs结合，包括MTK1、ASK1、TAK1、MEKK1和Raf-1。DVD位点位于MAPKK催化结构域的c端，由大约20个氨基酸组成。无论在体外还是体内，DVD位点的突变都强烈抑制MAPKKs与特异性MAPKKKs结合并被其激活。DVD位点突变体在体内不能被各种外部刺激激活。此外，在体外和体内，合成的DVD寡肽通过竞争性抑制DVD对接，有效地抑制特异性MAPKK的激活，表明DVD介导的相互作用可能是药物开发治疗th1介导的自身免疫性疾病的有效靶点。少

Abnormal activation of T helper type 1 (Th1) cells plays pathogenic roles in allergic reactions and autoimmune diseases. A key cytokine in mediating Th1 cell function and autoimmunity is IFNγ. We have previously cloned a human stress-responsive MAPKKK, MTK1, and its activators, GADD45B and γ, and showed that these molecules are indispensable for effector function of Th1 cells : Expression of GADD45β/γ is induced during Th1 cell differentiation, which in turn activates the p38 MAPK signaling pathway through MTK1 activation, and promotes IFNγ production in Th1 cells. The aims of this study were 1) to elucidate regulatory mechanisms of GADD45-induced MTK1 activation and 2) to develop novel methods to inhibit MTK1-p38 signaling.1) We dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus binds to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction is disrupted by the binding of GADD45 to the MTK1 N-terminal GAD … More D45-binding site. GADD45 binding also induced MTK1 dimerization via a domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. We thus conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain.2) We found a conserved docking site, termed DVD, in the mammalian MAPKKs belonging to the three major subfamilies, namely MEK1/2, MKK4/7, and MKK3/6. The DVD sites bind to their specific upstream MAPKKKs, including MTK1, ASK1, TAK1, MEKK1, and Raf-1. The DVD site is a stretch of about 20 amino acids immediately on the C-terminal side of the MAPKK catalytic domain. Mutations in the DVD site strongly inhibited MAPKKs from binding to, and being activated by, their specific MAPKKKs, both in vitro and in vivo. DVD site mutants could not be activated by various external stimuli in vivo. Moreover, synthetic DVD oligopeptides efficiently inhibited specific MAPKK activation, both in vitro and in vivo, through competitive inhibition of DVD docking, demonstrating that the DVD-mediated interaction may be an effective target for drug development to treat Th1-mediated autoimmune diseases. Less