Regulation of SIGNALLING PATHWAYS INVOLVING NUCLEAR FACTOR KAPPA B

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Abstract

Regulation of genes for several lymphokines 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, is 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. We have discovered a rare clinical condition of immunodeficiency and loss of lymphocyte homeostasis due to an inherited genetic deficiency of caspase-8. Remarkably, our studies revealed that the immunodeficiency was caused by a defect in NF-kappaB induction in lymphocytes following antigen activation. In particular, we found that caspase-8 played a direct role in physically linking the membrane-associated protein complex containing the Carma 1, MALT 1, and the Bcl-10 proteins to the I-kappaB kinase complex. Furthermore, the proteolytic enzyme activity of caspase-8 appears to be crucial for it to convey the activation signal. Precisely how this occurs, especially the identity of the hypothetical proteolytic substrate of caspase-8, is under investigation. Additional examples of caspase-8 deficiency are being sought in the human population to understand the full clinical and biochemical manifestations of this genetic defect. In exploring the role of caspase-8, we discovered a vital role for a 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 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. [unreadable] While studying the induction of NF-kappaB in lymphocytes, we have found a third component of the complex which has proved to be crucial to the gene induction capabilities of this transcription factor. The novel component associates with critical subunits of the complex and elimination of the third component potently inhibits the capability of NF-kappaB to mediate gene induction despite the fact that it has no effect on shuttling of the transcription complex from the cytoplasm to the nucleus. Further biochemical characterization of the third component, which we provisionally named A-kappaB for activator of kappaB, is presently being carried out. Remarkably, this novel component is crucial for regulating a subset of NF-kappaB regulated genes,including the immunoglobulin kappa light chain gene and the IL-2 gene, but not all such genes. Given its pivotal role in transcriptional activation, this implies that there will be other similar factors to control the remaining NF-kappaB-regulated genes and we are searching for such a factor(s). 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.