Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cellfree system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation.Ras GTPases (Ha-, Ki-, and N-Ras) are the key proteins in eukaryotic signal transduction directed toward cellular proliferation and differentiation (1-3). The biological activity of Ras is controlled by a regulated GDP/GTP cycle. Guanine-nucleotide exchange factors (Ras-GRF1/2, mSos1/2) induce dissociation of GDP from Ras⅐GDP to form an active, GTP-bound form of Ras. On the other hand, GTPase-activating proteins (p120GAP, NF1) accelerate the intrinsic GTP hydrolytic activity of Ras to promote the formation of an inactive, GDP-bound form of Ras. Upon binding of GTP, Ras alters its conformation to interact with multiple downstream effectors. One of the well characterized effectors is a serine/threonine kinase Raf-1 (4, 5), which induces activation of a dual specificity kinase MEK.1 Activated MEK in turn activates a serine/threonine kinase ERK, which phosphorylates a variety of proteins including protein kinases, transcription factors, and cytoskeletal proteins (6).Although it has been demonstrated that Ras binds to Raf-1 directly, the precise mechanism of Raf-1 activation by Ras is not fully understood. According to the current model (5), when Ras is activated, the effector region of Ras interacts with the Ras binding domain of Raf-1, which leads to the binding of cysteine-rich domain of Raf-1 to Ras. These interactions relieve the masking of the C-terminal catalytic domain of Raf-1 by the N-terminal regulatory domain and allow Raf-1 to interact with the downstream kinase, MEK. However, since direct interaction of Ras with Raf-1 is insufficient for Raf-1 activation (5, 7), an additional molecule(s) has been expected to be involved in this activation. In fact, using a cell-free system, we have found a Ras-dependent Ra...

The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the basic helix-loop-helix-PER-ARNT-SIM superfamily. Xenobiotics, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, bind the receptor and trigger diverse biological reactions. Thymocyte development and T cell-dependent immune reactions are sensitive targets of AhR-dependent 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity. However, the exact role of the AhR in T cells in animals exposed to exogenous ligands has not been clarified because indirect effects of activated AhR in other cell types cannot be excluded. In this study, we generated transgenic (Tg) mice expressing a constitutively active mutant of AhR under the regulation of a T cell-specific CD2 promoter to examine AhR function in T cells. The mRNAs of the constitutively active mutant of AhR and an AhR-induced gene, CYP1A1, were expressed in the thymus and spleen of the Tg mice. The transgene expression was clearly detected in the thymocytes, CD4, and CD8 T cells, but not in the B cells or thymus stromal cells. These Tg mice had a decreased number of thymocytes and an increased percentage of CD8 single-positive thymocytes, but their splenocytes were much less affected. By contrast, the increase in number of T cells and B cells taking place in the spleen after immunization was significantly suppressed in the Tg mice. These results clearly show that AhR activation in the T-lineage cells is directly involved in thymocyte loss and skewed differentiation. They also indicate that AhR activation in T cells and not in B cells suppresses the immunization-induced increase in both T cells and B cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress antigen-specific antibody production in humoral immune reactions, but the precise mechanism remains unclear. Since T cell activation and subsequent production of type 2 helper T (Th2) cell-derived cytokines are required for antigen-specific antibody production in humoral immunity, we examined the effects of TCDD on splenic T cell numbers, T cell growth factor IL-2 production, and Th2 cell-derived cytokine production. C57BL/6N mice were orally given TCDD (20 microg/kg) or vehicle, and immediately intraperitoneally immunized with ovalbumin (OVA) adsorbed to alum, and cellular changes in the spleen and cytokine production by spleen cells were investigated from Day 1 to Day 14. In vehicle-control mice the numbers of splenic CD3(+) T cells increased from Day 7 onward, but no increase was observed in the TCDD-exposed mice. When spleen cells from control mice were cultured and restimulated with OVA ex vivo, a significant amount of IL-2 was found from Day 1, but it decreased on Day 7, whereas TCDD exposure promptly suppressed the increase on Day 4. TCDD exposure significantly suppressed the production of Th2 cell-derived cytokines IL-4, IL-5, and IL-6, which were prominently increased from Day 4 onward in control mice. The dose-dependent study showed that IL-5 production was significantly suppressed in a dose-dependent manner starting at 1 microg/kg TCDD. Moreover, separation and reconstitution studies showed that the TCDD-induced suppression of IL-5 production was due to the impaired function of T cells rather than that of antigen-presenting cells. The results of this study suggest that TCDD-induced suppression of T cell activation and Th2-type cytokine production is involved in the impairment of antigen-specific antibody production.

No abstract