Regulation of SIGNALLING PATHWAYS INVOLVING NUCLEAR FACTOR KAPPA B

National Institute Of Allergy And Infectious Diseases

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Abstract

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. However, this transcription factor plays a role in the induction of diverse sets of genes throughout the body in response to hundreds of different inducers. Overall there are 5 Rel protein subunits, including p50 and p65, that form a variety of hetero- or homodimers that have been widely studied and have important roles in the immune system. Nevertheless, very little is known about the kinetics of NF-kB dimer formation or the stability or interconversion of dimerised Rel subunits. We therefore carried out a quantitative analysis of association and disassociation properties of purified p50 and p65 Rel subunits using light scattering, mass spectrometry, and other biophysical techniques. Our data show that at physiological temperatures, p50 or p65 homodimers efficiently exchange subunits with a half-life of less than 10 minutes. Even more interesting, we found a marked preference for the formation of the p50/p65 heterodimer, which kinetic analyses show was at least 10 times more stable than either homodimer. The implications of these findings are that specific DNA targets of either the p50 or p65 homodimers may be restricted to situations in which these subunits are expressed exclusively, or require additional chromatin factors or post-translational modifications. Together, our work indicates that cells can modulate NF-kB activity by finely modulating the relative proportions of the p50 and p65 proteins, which will determine the quantities of different homo- or hetero-dimers. This work thus provides a new quantitative understanding of Rel dimer distribution in the cell, which has important implications for our understanding of how this transcription factor system controls genes in the healthy and diseased immune system.