Regulation of SIGNALLING PATHWAYS INVOLVING NUCLEAR FACTOR KAPPA B

许多免疫反应基因的调节依赖于一个称为kappaB的10个碱基对的DNA序列。这个序列被一系列与rel癌基因相关的蛋白质因子结合在一起。与该序列结合的原型转录复合体称为NF-kappaB，通常被定义为p50 DNA结合蛋白和p65(RelA)激活蛋白之间的异源二聚体，后者通常被一种称为I-kappaB的蛋白隔离在细胞质中。在对细胞进行某些类型的刺激后，一种称为I-kappaB的特殊蛋白激酶复合体导致I-kappaB的磷酸化，随后它被泛素化和降解。在可以释放核因子-kappaB的刺激中，有一种是在免疫反应中由抗原触发T细胞受体(TCR)或B细胞受体(BCR)。在研究一种罕见的免疫缺陷的临床情况时，我们发现核因子-kappaB具有比先前怀疑的更复杂的亚基组成。具体地说，我们发现IL-2和I-kB等基因含有特定序列的kappaB位点，该位点结合了包含p50、p65和核糖体蛋白小亚基3(RPS3)的三聚体复合体。RPS3是一种含有K-同源(KH)结构域的蛋白质，它与单链核酸结合，并显著增强p50和p65同源蛋白与这些选定的kappaB位点的亲和力。核因子-kappaB基因的一部分依赖于RPS3，包括重要的生理功能，如免疫球蛋白kappaB轻链基因在B细胞中的表达和IL-2基因在T细胞中的表达。我们进一步探索了这一新的核因子-kappaB基因调控范式，以潜在地解释含有不同版本的核因子-kappaB结合位点的基因的选择性激活。这导致我们寻找也可能参与结合复合体的其他亚基。我们已经确定，存在一个独特的KH结构域蛋白，称为wan23，它是NF-kappaB复合体的一个关键亚基，与CD25(IL-2受体阿尔法链)基因启动子中发现的特定kappaB结合位点结合。DNA结合分析表明，wan23显著增强了p50和p65rel亚基与CD25启动子位点的结合。此外，如果wan23被siRNA耗尽，则由核因子-kappaB介导的该位点的转录活性受到严重抑制。在功能分析中，我们已经确定wan23参与了核因子-kappaB介导的人类白血病细胞系的基因调控，从而控制了这些细胞在细胞培养中对白细胞介素2的反应。如果我们降低wan23的表达，各种人类白血病细胞系如Hut-102或MT-2的存活率都明显降低。因此，wan23是特定的核因子-kappaB调控复合体的关键生理成分。这验证了我们的假设，即存在一组具有不同亚基组成的核NF-kappaB复合体，这些亚基以前被视为单一的NF-kappaB复合体。这些发现阐明了核因子-kappaB介导特定基因调控效应的基本机制。进一步的工作是为了了解RPS3和wan23KH结构域蛋白是如何被调控成在特定基因处并入核因子-kappaB复合体的。初步分析表明，特定的磷酸化事件在这一过程中发挥了关键作用。我们还试图确定这一成分的抑制剂，因为抑制核因子-kappaB是许多炎症和退行性疾病的主要治疗靶点。抑制核因子-kappaB也可能对涉及病原体的各种传染病有用，例如艾滋病毒，这些病原体利用这种因子的生命周期或致病效应。特别是，我们正在研究与HIV长末端重复转录启动子中的kappaB位点结合的NF-kappaB复合体的亚基组成。 我们也一直在研究在触发B细胞或T细胞上的抗原受体后诱导核因子-kappaB的激活电路。我们已经发现了一种新的激酶，它在物理上将包含Carma 1、MALT 1和Bcl-10(CBM)蛋白的膜相关蛋白复合体与I-kappaB激酶复合体连接起来。我们已经证明了细胞激酶在诱导途径中起着至关重要的作用。这种新的激酶既有正向调节作用，也有负向调节作用，它将来自淋巴细胞表面抗原受体的信号传递到基因诱导装置。该激酶已被证明参与发育和昼夜节律途径，现在似乎在免疫功能中发挥关键作用。我们将研究其调节的生化特征，以了解它可能如何在免疫系统的各种疾病中调节。质谱分析还揭示了涉及CBM复合体的信号转导途径中的其他蛋白质，实验将被用于阐明这些蛋白质的功能作用。

Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a P50 DNA binding protein and a P65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. While studying a rare clinical condition of immunodeficiency, we discovered that NF-kappaB has a more complex subunit composition than previously suspected. Specifically, we found that genes such as IL-2 and I-kB harbor kappaB sites of a particular sequence that bind a trimeric complex containing p50, p65, and ribosomal protein small subunit 3 (RPS3). RPS3 is a K-homology (KH) domain-containing protein that binds single-stranded nucleic acids and dramatically enhances the affinity of the p50 and p65 Rel-homology proteins to these select kappaB sites. A subset of all NF-kappaB genes are dependent on RPS3 including crucial physiological functions such as expression of the immunoglobulin kappa light chain gene in B cells and the interleukin-2 gene in T cells. We have further explored this new paradigm for NF-kappaB gene regulation to potentially explains the selective activation of genes harboring distinct versions of NF-kappaB binding sites. This has led us to search for other subunits that may also participate in the binding complex. We have determined that there is a distinct KH domain protein called wan23 is a critical subunit of the NF-kappaB complex that binds to the particular kappaB binding site found in the promoter of the CD25 (the IL-2 receptor alpha chain) gene. DNA-binding analyses reveal that wan23 dramatically enhances the binding of the p50 and p65 Rel subunits to the CD25 promoter site. Moreover, the transcriptional activity of this site mediated by NF-kappaB is profoundly suppressed if wan23 is depleted by siRNA. In functional analyses, we have determined that the participation of wan23 in NF-kappaB-mediated gene regulation in human leukemia cell lines governs the survival of these cells in response to interleukin-2 in cell culture. If we reduce the expression of wan23, various human leukemia cell lines such as Hut-102 or MT-2 have markedly reduced viability. Therefore, wan23 is a critical physiological component of particular NF-kappaB regulatory complexes. This has verified our hypothesis that there are a group of nuclear NF-kappaB complexes that have distinct subunit compositions that have previously been viewed as a single NF-kappaB complex. These discoveries elucidate a fundamental mechanism by which NF-kappaB mediates specific gene regulatory effects. Further work is being directed to understanding how the RPS3 and wan23 KH domain proteins are regulated to become incorporated into the NF-kappaB complexes at specific genes. Preliminary analyses suggest that specific phosphorylation events play a key role in this process. We are also attempting to identify inhibitors of this component, since inhibition of NF-kappaB is a prime therapeutic target for number of inflammatory and degenerative conditions. Inhibition of NF-kappaB may also be useful for various infectious diseases involving pathogens, such as HIV, that utilize this factor for their life cycle or pathogenic effects. In particular, we are studying the subunit composition of the NF-kappaB complex that binds to the kappaB sites in the HIV long terminal repeat transcriptional promoter. We have also been studying the activation circuitry that induces NF-kappaB after triggering the antigen receptor on B cells or T cells. We have found a new kinase that plays a direct role in physically linking the membrane-associated protein complex containing the Carma 1, MALT 1, and the Bcl-10 (CBM) proteins to the I-kappaB kinase complex. We have demonstrated a vital role for the cellular kinase in the induction pathway. This novel kinase has both a positive and negative regulatory role is transducing the signals from antigen receptors at the surface of lymphocytes to the gene induction apparatus. This kinase has been shown to be involved in developmental and circadian rhythm pathways and now appears to play a key role in immune function. We will be studying the biochemical features of its regulation to understand how it might be modulated in various diseases of the immune system. Mass spectrometry analysis has also revealed other proteins in the signal transduction pathway involving the CBM complex and experimentation will be directed to elucidating the functional role of these proteins.