GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion.

The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.     

活化的T细胞核内因子（NFAT）的调节及其在神经系统中的功能

活化的T细胞核内因子（nuclear factor of activated T-cells, NFAT）作为细胞信号转导通路中的一类重要的转录因子参与细胞功能的调节. NFAT的活化主要是通过细胞内钙/钙调神经磷酸酶（Ca2+/calcineurin）的刺激启动,它脱磷酸后发生核转位并与DNA的特定序列结合,同时通过与其它转录因子的协同作用,调节目的基因的特定表达. NFAT在免疫系统中所调节的基因表达已经得到了充分的研究. 近年实验研究发现,NFAT的转录因子家族在脊椎动物的神经系统中也发挥着非常重要的作用. 本文综述了NFAT家族蛋白的分类、结构、磷酸酶与激酶对其出入核的调节及在神经系统中的研究进展,使得能够更加全面地认识calcineurin/NFAT信号通路的作用. 此外,由于环孢菌素A（cyclosporin A）等药物在神经系统应用的局限性,对于NFAT调节深入研究,也将为筛选或者开发更为高效、低毒药物提供新的思路.     

Reciprocal NFAT1 and NFAT2 nuclear localization in CD8+ anergic T cells is regulated by suboptimal calcium signaling

Anergy is an important mechanism of maintaining peripheral immune tolerance. T cells rendered anergic are refractory to further stimulation and are characterized by defective proliferation and IL-2 production. We used a model of in vivo anergy induction in murine CD8+ T cells to analyze the initial signaling events in anergic T cells. Tolerant T cells displayed reduced phospholipase Cgamma activation and calcium mobilization, indicating a defect in calcium signaling. This correlated with a block in nuclear localization of NFAT1 in anergic cells. However, we found that stimulation of anergic, but not naive T cells induced nuclear translocation of NFAT2. This suggested that NFAT2 is activated preferentially by reduced calcium signaling, and we confirmed this hypothesis by stimulating naive T cells under conditions of calcium limitation or partial calcineurin inhibition. Thus, our work provides new insight into how T cell stimulation conditions might dictate specific NFAT isoform activation and implicates NFAT2 involvement in the expression of anergy-related genes.     

Inhibition of drug-induced Fas ligand transcription and apoptosis by Bcl-XL.

Fas/Fas ligand system triggers apoptosis in many cell types. Bcl-XL overexpresion antagonizes Fas/Fas ligand-mediated cell death. The mechanism by which Bcl-XL influences Fas-mediated cell death is unclear. We have found that microtubule-damaging drugs (e.g. Paclitaxel) induce apoptosis in a Fas/FasL-dependent manner. Inhibition of Fas/FasL pathway by anti-FasL antibody, mutant Fas or a dominant negative FADD blocks paclitaxel-induced apoptosis. Paclitaxel induced apoptosis through activation of both caspase-8 and caspase-3. Overexpression of Bcl-XL leads to inhibition of paclitaxel-induced FasL expression and apoptosis. Bcl-XL prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes) by inhibiting the activation of calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT for it to move to the nucleus. The loop domain in Bcl-XL can suppress the anti-apoptotic function of Bcl-XL and may be a target for regulatory post-translational modifications. Upon phosphorylation, Bcl-XL loses its ability to bind with calcineurin. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, paclitaxel and other drugs that disturb microtubule function kill cells, at least in part, through the induction of FasL, and Bcl-XL-mediated resistance to these agents is related to failure to induce FasL expression.     

Calcineurin and NFAT4 induce chondrogenesis

Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Here we show that elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. This suggested that calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Furthermore, activated NFAT4 induced BMP2 gene expression. These results have important implications for the effects of NFATs during development and adaptive responses.