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

辅助性T细胞1型(Th1)的异常激活在变态反应和自身免疫性疾病中起致病作用。干扰素γ是调节Th1细胞功能和自身免疫的关键细胞因子。我们已经克隆了人类应激反应MAPKKK1及其激活子GADD45B和γ，并证明这些分子对Th1细胞的效应功能是必不可少的：GADD45β/γ的表达是在Th1细胞分化过程中诱导的，进而通过MTK1激活激活p38MAPK信号通路，促进Th1细胞产生干扰素γ。本研究的目的是：1)阐明GADD45诱导MTK1激活的调控机制；2)寻找抑制MTK1-p38信号转导的新方法。1)剖析GADD45蛋白激活MTK1的分子机制。MTK1的N末端与其C-末端片段结合，从而抑制C-末端的激酶域。这种N-C相互作用被GADD45与MTK1N端GAD…的结合所破坏更多的D45结合位点。GADD45结合还通过一个包含卷曲螺旋基序的结构域诱导MTK1二聚，这对于在激酶激活环中Thr-1493处MTK1的反式自动磷酸化是必不可少的。因此，我们得出结论：GADD45结合诱导MTK1N-C解离、二聚化和Thr-1493处的自动磷酸化，导致激酶催化结构域的激活。2)我们在哺乳动物的MAPKK中发现了一个保守的对接位点，称为DVD，属于三个主要亚家族，即MEK1/2，MKK4/7和MKK3/6。DVD端位于MAPKK催化结构域的C-末端，由大约20个氨基酸组成。在体外和体内，DVD位点的突变强烈地抑制MAPKKs与其特定的MAPKKs结合并被其激活。在体内，DVD位点突变体不能被各种外界刺激激活。此外，合成的DVD寡肽通过竞争性抑制DVD对接，在体外和体内都有效地抑制了特异性MAPKK的激活，表明DVD介导的相互作用可能是治疗Th1介导的自身免疫性疾病的药物开发的有效靶点。较少

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