Molecular mechanism of signal transduction mediated by Rel/NFkB

1.Rel/NFkappaB激活的分子机制REL/NFkappaB的激活是通过信号诱导抑制蛋白IkappaBalpha的快速磷酸化和降解来实现的。为了探索信号诱导IkappaBalpha耗尽的分子机制，我们描绘了IkappaBalpha中调控所需的结构域。与以前的报告不同，害虫类序列对于TNFpha诱导的降解是必不可少的，而六个锚定蛋白重复是必需的。然而，包括PEST类序列在内的锚蛋白重复序列和羧基末端区域都不是肿瘤坏死因子α诱导的磷酸化所必需的。从膜受体到Rel/IkappaBalpha复合体的信号转导我们发现，CD40发出的信号与Rel/NFkappaB的激活有关。为了确定转导CD40信号的分子，我们利用酵母双杂交系统克隆了编码CD40胞浆尾部结合蛋白的cDNA，编码一个可能的信号转导蛋白的cDNA被分子克隆，命名为TRAF5和TRAF6。TRAF5或TRAF6的过表达激活了转录因子NFkappaB，因此，我们正在寻找这些蛋白的下游转导因子。此外，我们还证实了铜离子通过抑制信号诱导的IkappaBalpha靶分子的磷酸化来阻断NFkappaB的激活。新的kappaB蛋白的鉴定为了鉴定新的kappaB cDNAs，以NFkappaB的p65亚基为探针，筛选了一个表达文库。本研究仍在进行中。Rat中Rel、NFkappaB和IkappaB基因的染色体定位我们已确定NFKB-1(p50，IkappaBGamma)位于大鼠的2号染色体上，Rel和IkappaBalpha的定位仍在进行中。

1. Molecular Mechanism of Rel/NFkappaB activationActivation of Rel/NFkappaB is achieved by the signal-induced rapid phosphorylation and degradation of inhibitory protein IkappaBalpha. To explore the molecular mechanism of signal-induced depletion of IkappaBalpha, we have delineated the domain in IkappaBalpha that is required for the regulation. In contrast to the previous reports, the PEST-like sequences dispensable for TNFalpha-induced degradation whereas the six ankyrin repeats are required. However, neither ankyrin repeats nor carboxyl-terminal region including the PEST-like sequences are required for TNFalpha-induced phosphorylation.2. Signal transduction pathway emanating from membrane receptors to Rel/IkappaBalpha complexWe have found that signals emanating from CD40 link to Rel/NFkappaB activation. To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40, cDNAs encoding a putative signal transducer protein, designated TRAF5 and TRAF6, have been molecularly cloned. Overexpression of TRAF5 or TRAF6 activates transcription factor NFkappaB.Thus, we are looking for the down stream transducer of these proteins. Furthermore we have demonstrated that cupric ion blocks NFkappaB activation through inhibiting the signal-induced phosphorylation of IkappaBalpha Target molecule of cupric ion remain to be identified.3. Identification of novel kappaB proteinsTo identify novel IkappaB cDNAs, an expression cDNA library has been screened using p65 subunit of NFkappaB as a probe. This study is still in the progress.4. Chromosomal assignment of rel, NFkappaB and IkappaB genes in RatWe have indentified that NFKB-1 (p50, IkappaBgamma) in on the Rat chromosome #2. Assignment of rel and IkappaBalpha is still in the Progress.