Regulation of SIGNALLING PATHWAYS INVOLVING NUCLEAR FACTOR KAPPA B

许多免疫应答基因的调节依赖于称为kappaB的10 bp DNA序列。该序列被与Rel癌基因相关的蛋白质因子家族结合。 与该序列结合的原型转录复合物，称为NF-κ B，通常被定义为P50 DNA结合蛋白和P65（RelA）活化蛋白之间的异二聚体，其通常被称为I-kappaB的蛋白质隔离在细胞质中。在对细胞进行某些类型的刺激后，一种称为I-kappaB激酶的特异性蛋白激酶复合物引起I-kappaB的磷酸化，随后是其泛素化和降解。在能够释放NF-κ B的刺激物中，有一种是在免疫应答过程中抗原对T细胞受体（TCR）或B细胞受体（BCR）的触发。 在研究一种罕见的免疫缺陷临床状况时，我们发现NF-κ B具有比以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等基因具有特定序列的kappaB位点，该位点结合含有p50、p65和核糖体蛋白小亚基3（RPS 3）的三聚体复合物。 RPS 3是一种含有K-同源（KH）结构域的蛋白，其结合单链核酸并显著增强p50和p65 Rel-同源蛋白对这些选择的kappaB位点的亲和力。 所有NF-κ B基因的一个子集依赖于RPS 3，包括关键的生理功能，如免疫球蛋白κ轻链基因在B细胞中的表达和白细胞介素-2基因在T细胞中的表达。 我们进一步探索了NF-κ B基因调控的新模式，以潜在地解释携带不同版本NF-κ B结合位点的基因的选择性激活。这促使我们寻找其他可能参与结合复合物的亚基。我们已经确定，有一个独特的KH结构域蛋白称为wan 23是NF-κ B复合物的关键亚基，结合到CD 25（IL-2受体α链）基因启动子中发现的特定κ B结合位点。DNA结合分析表明，wan 23显着增强的p50和p65 Rel亚基的CD 25启动子位点的结合。 此外，如果wan 23被siRNA耗尽，则由NF-κ B介导的该位点的转录活性被深度抑制。在功能分析中，我们已经确定，在人白血病细胞系中，参与NF-κ B介导的基因调控的wan 23控制这些细胞在细胞培养中对白细胞介素-2的应答中的存活。如果我们降低wan 23的表达，各种人白血病细胞系如Hut-102或MT-2的生存力显著降低。因此，wan 23是特定NF-κ B调节复合物的关键生理组分。这证实了我们的假设，即有一组核NF-κ B复合物，具有不同的亚基组成，以前被视为一个单一的NF-κ B复合物。这些发现阐明了NF-κ B介导特定基因调控作用的基本机制。进一步的工作是针对了解如何RPS 3和wan 23 KH结构域蛋白被调节，成为纳入NF-κ B复合物在特定的基因。 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。我们还试图确定该组分的抑制剂，因为抑制NF-κ B是许多炎症和退行性疾病的主要治疗靶点。 NF-κ B的抑制也可用于涉及病原体的各种感染性疾病，例如HIV，其利用该因子用于其生命周期或致病作用。 特别是，我们正在研究NF-κ B复合物的亚基组成，该复合物与HIV长末端重复转录启动子中的κ B位点结合。 我们也一直在研究触发B细胞或T细胞上的抗原受体后诱导NF-κ B的激活电路。 我们已经发现了一种新的激酶，它在将包含Carma 1、MALT 1和Bcl-10（CBM）蛋白的膜相关蛋白复合物与I-kappaB激酶复合物物理连接中起直接作用。 我们已经证明了细胞激酶在诱导途径中的重要作用。 这种新的激酶具有正性和负性调节作用，将信号从淋巴细胞表面的抗原受体传递到基因诱导装置。 这种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，以了解它如何在各种免疫系统疾病中被调节。 质谱分析还揭示了涉及CBM复合物的信号转导途径中的其他蛋白质，实验将致力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.