RAF ACTIVATES NF-KB DRIVEN EXPRESSION VIA THE ACTIVATION OF GABP

Raf-1是一种丝氨酸/苏氨酸蛋白激酶，其在细胞凋亡中起核心作用。 从细胞膜到细胞核的信号转导。 激活的Raf-1 激活AP-1、Ets和NF-B结合位点的转录。 拉夫 诱导人NF-~B结合位点的反式激活 免疫缺陷病毒（HIV）长末端重复序列（LTR）似乎导致 来自Raf-activating GABP，一种ets家族转录因子， 由两个亚基（α和β）组成。 GABP过表达 诱导的HIV LTR表达和活化的Raf与 GABP用于HIV-LTR驱动的表达。 GABP α突变体携带 磷酸化位点和IkappaB中的单个氨基酸取代 α似乎降低了协同转录活性。 在 相反，两种不同GABP β磷酸化的点突变 位点似乎对协同活性没有影响。 使用凝胶 移位分析，我们已经确定了纯化的GABP与NF-κ B结合 在体外的HIV-LTR的结合位点。 这种结合是特异性的， 需要两个NF-κ B结合位点，因为单个位点不能 竞争约束力。 GABP α和GABP β似乎在 在体内Raf活化后的异二聚体复合物， 免疫共沉淀实验。 为了确定激活的拉夫 直接磷酸化GABP，我们使用由混合物产生的活化Raf 用表达Raf-1、v-Ras和 好吧 尽管这种激活的Raf有效地磷酸化了丝裂原， 激活的蛋白质（MAP）激酶激酶启动激酶级联， 导致Jun被MAP激酶磷酸化，直接GABP 未观察到磷酸化。 然而，激活的MAP激酶 在体外磷酸化GABP α和β 1亚基。 此外，委员会认为， 激活的Raf-1似乎诱导GABP β 1的磷酸化， vivo.

Raf-1 is a serine/threonine protein kinase that plays a central role in signal transduction from the membrane to the nucleus. Activated Raf-1 activates transcription from AP-1, Ets, and NF-~B binding sites. Raf- induced transactivation from the NF-~B binding sites in the human immunodeficiency virus (HIV) long terminal repeat (LTR) appears to result from Raf-activating GABP, an ets family transcription factor that is composed of two subunits (alpha and beta). Overexpression of GABP induced expression from the HIV LTR and activated Raf synergized with GABP for HIV-LTR-driven expression. The GABP alpha mutant carrying a single amino acid substitution in the phosphorylation site and IkappaB alpha appear to reduce synergistic transcriptional activity. In contrast, point mutations in two different GABP beta phosphorylation sites appear to have no effect on synergistic activity. Using a gel shift assay, we have determined that purified GABP binds to the NF-kappaB binding sites in the HIV-LTR in vitro. This binding was specific and required both NF-kappaB binding sites since a single site failed to compete for binding. GABP alpha and GABP beta appear to associate in a heterodimeric complex after Raf activation in vivo as judged by co-immunoprecipitation experiments. To determine if activated Raf phosphorylates GABP directly, we used activated Raf produced by mixed infections in Sf9 cells with baculoviruses expressing Raf-1, v-Ras and Lck. Although this activated Raf efficiently phosphorylated mitogen- activated protein (MAP) kinase kinase initiating a kinase cascade which resulted in Jun phosphorylation by MAP kinase, direct GABP phosphorylation was not observed. Activated MAP kinase, however, did phosphorylate GABP alpha and beta1 subunits in vitro. Moreover, activated Raf-1 appears to induce the phosphorylation of GABP beta1 in vivo.