Zhijian James Chen
NF-kB signaling and the ubiquitin-proteasome pathway
The transcription factor NF-kB regulates the expression of a large array of genes involved in
various physiological and pathological processes, including inflammation, immune responses,
viral pathogenesis, and apoptosis. The function of NF-kB is tightly regulated through interactions with inhibitory proteins termed IkB (ie., IkBa), which sequester NF-kB in the cytoplasm of quiescent cells. When cells are stimulated with NF-kB agonists, IkBa is rapidly phosphorylated and degraded, thereby releasing NF-kB which then enters the nucleus to turn on its target genes.
Our laboratory is interested in understanding the molecular mechanism by which IkB proteins are phosphorylated and degraded, as these are the key regulatory steps in the NF-kB signaling pathway. Our main approach is to establish an inducible cell-free system for the phosphorylation and degradation of IkBa, and then purify and clone various enzymes or factors involved in the process. By using okadaic acid-supplemented HeLa cell cytoplasmic extracts, we demonstrated that IkBa is degraded via the ubiquitin-proteasome pathway, and ubiquitination is regulated by site-specific phosphorylation. We further identified a large (~700 kDa) protein kinase complex capable of phosphorylating IkBa at specific sites. Surprisingly, this kinase can be activated independently by ubiquitination as well as by phosphorylation. Thus, IkB kinase may serve as a signal integrator that translates information from diverse signaling pathways into NF-kB activation.
Current research activities are focused on: 1) molecular characterization of the IkB kinase complex; 2) understanding the mechanism of IkB kinase activation, especially the novel mechanism of ubiquitin-dependent activation of the kinase activity; 3) identification and characterization of the enzymatic machinary responsible for the ubiquitin-dependent degradation of IkB. A variety of molecular, cellular and biochemical techniques are used in these studies. The overall goal is to understand gene regulation by phosphorylation, ubiquitination, and proteolysis, and to eventually exploit knowledge gained from these studies for the treatment of human diseases.