| Budget Amount *help |
¥15,300,000 (Direct Cost: ¥15,300,000)
Fiscal Year 2006: ¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 2005: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Research Abstract |
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
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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
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