Insights into the homeostatic regulation of I kappa B alpha

The regulation of the transcription factor NF-kappaB is crucial to proper cell physiology, as misregulation of this transcription factor can lead to many disease states, including chronic inflammation and cancer. NF-kappaB is inhibited by a class of inhibitor proteins known as IkappaB; the most effective IkappaB is IkappaBalpha. Signal induced degradation of IkappaBalpha leading to NF-kappaB translocation and activation is well documented and requires post-translational modifications such as phosphorylation and ubiquitination. It has recently been demonstrated that IkappaBalpha also undergoes stimulus-independent degradation and this degradation pathway might be important for NF-kappaB activity regulation. The focus of this study is to investigate the mechanism of stimulus independent degradation of IkappaBalpha and its effect on NF-kappaB activity. Chapter 1 introduces the NF-kappaB:IkappaB signaling system, ubiquitin-independent degradation of several substrates, and also various regulatory proteasome complexes. Chapter 3 describes the delineation of the pathways regulating the degradation of IkappaBalpha. Results presented here show that the degradation pathway of IkappaBalpha is determined by binding to NF-kappaB subunits. It is further shown that perturbations of ubiquitin-independent degradation pathway alter NF-kappaB activation. Chapter 4 focuses on the ankyrin repeat sequence of IkappaBalpha. Of the six ankyrin repeats present in IkappaBalpha, several deviate from the consensus ankyrin repeat sequence. Mutations back to the consensus sequence in several ankyrin repeats demonstrate that the location of thermodynamic stabilization determines the degradation rate of IkappaBalpha. Finally, Chapter 5 dissects the degradation requirements of IkappaBalpha. Our results show that there are two degrons within IkappaBalpha; one located in the 5th ankyrin repeat, and the other within the PEST domain of IkappaBalpha. Both degrons are controlled by hydrophobic residues within long stretches of flexible regions.