Degradational and Transcriptional Investigations of the RAF Kinase Inhibitory Protein

RKIP (Raf-1 kinase inhibitory protein) is a novel potent metastasis suppressor by its function of inhibiting ERK pathway, NF-κB pathway, and GRK-2. RKIP expression level is reported to be critical in the differentiation process and the pathogenesis of many different cancers and inflammatory diseases. However, the understanding of how RKIP expression level is regulated is poor. In this thesis, I endeavored to investigate whether RKIP is regulated by degradational mechanisms using ubiquitination and cycloheximide blocking experiments or transcriptional mechanisms other than by Snail repression using luciferase reporter assays. My results indicate that RKIP is a relatively stable protein, which has a half-life around 1 to 2 days and is not subject to rapid degradation by ubiquitin-proteosomal pathway in either human HEK293 cells or rat AR42J cells. I found that the RKIP promoter reporter was not significantly repressed by Snail in transfected HEK293 cells but was subject to repression by the GSK-3 inhibitor BIO compound in both E-box (putative Snail binding site)-dependent and -independent manner. My results indicate that there are other Snail-independent transcriptional regulations involved in the regulation of RKIP expression.