|Ph.D Student||Brunwasser Michal|
|Subject||Studying Looping-Based Transcriptional Regulation Using|
Synthetic Biology Tools
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Roee Amit|
|Full Thesis text|
The strength and duration of intracellular signals must be precisely regulated, since inappropriate signaling can cause disease. Negative feedback mechanisms provide an effective mean of controlling intracellular signals. Sef proteins are feedback antagonists of receptor tyrosine kinase signaling. In our group we have discovered a previously unknown function of human Sef (hSef-a) as a feedback antagonist of proinflammatory cytokine signaling. Sef is an early cytokine responsive gene whose expression is induced by NF-kB and AP-1 transcription factors, two key mediators of proinflammatory cytokine signaling. Sef in turn, inhibits transcriptional activation of NF-kB but not AP-1. The NF-kB family consists of 5 members including p65 and p50 which form transcriptionally active or repressive NF-kB dimers. In resting cells, classic NF-kB is found in the cytoplasm, bound to inhibitor of NF-kB termed IkBα. Ligand stimulation leads to activation of the IKK complex, which phosphorylates IkBα, sending it to degradation. This enables the nuclear translocation ofNF-kB. The goal of this work was to study how hSef-a exerts its inhibition of NF-kB signaling at the biochemical and cellular level. In IL-1 stimulated cells, ectopic hSef-a had no effect on the phosphorylation levels of Jun N-terminal kinase, required for AP-1 activation, confirming that hSef-a does not inhibit IL-1 induced AP-1 activation. In addition, hSef-a had no effect on IL-1 induced IKKα/β phosphorylation or IkBα degradation suggesting that hSef-a does not interfere with upstream components of the NF-kB signaling. This was confirmed by showing that hSef-a inhibits NF-kB reporter activity in the presence of constitutively active forms of IKKα or IKKβ. Immunofluorescence staining of endogenous p65 revealed that ectopic hSef-a inhibits IL-1 induced nuclear translocation of NF-kB. The effects observed with ectopic Sef were validated in Sef knockout cell line, which resulted in enhanced nuclear accumulation and transcriptional activity of NF-kB. In cells co-expressing hSef-a and p65, p65 co-localized with hSef-a. A point mutation in hSef-a (Y330F) abolished co-localization with p65 and inhibition of IL-1 induced NF-kB activation, implying that Sef and p65 interaction is crucial for Sef inhibitory function. This work revealed the existence of a novel regulatory mechanism that controls NF-kB nuclear translocation. Furthermore, it highlights Sef as a modulator of signals emanating from divergent receptor families. Since Sef emerges as a tumor suppressor gene, its involvement in cytokines signaling points to additional mechanisms by which hSef inactivation in cancer can contribute to the malignant process.