Identification of IkappaB kinase phosphorylation sites on human transcription factor REL and characterization of histone acetyltransferase P300 as a REL coactivator.

摘要

The NF-kappaB family of transcription factors is involved in the regulation of many cellular processes including inflammation, innate immunity, apoptosis, and cell growth and proliferation. One member of this family, human c-Rel (REL), is encoded by an oncogene that is amplified or otherwise altered in several types of human B-cell lymphoma. Overexpression of REL can also oncogenically transform chicken spleen cells and a human B-lymphoma cell line. This transforming activity of REL requires the activity of at least one of the two transcriptional activation subdomains in the C-terminal half of REL. The goal of this research is to understand further REL-mediated transactivation by investigating post-translational modification and protein-protein interactions involving REL transactivation domain sequences.;Although REL is primarily regulated by nuclear-cytoplasmic translocation through interaction with IkappaB inhibitors, REL undergoes post-translational modifications that have been proposed to modulate its transcriptional activation ability. Deletion analysis, site-directed mutagenesis, and immune complex kinase assays have been used to identify Ser484 and Ser494 as the primary sites of IkappaB kinase (IKK)-mediated in vitro phosphorylation in the C-terminal transactivation domain of REL. However, co-transfection studies failed to detect IKK-mediated phosphorylation of these sites in vivo, and mutation of Ser484 and Ser494 did not affect IKK's ability to enhance GAL4-REL transactivation. Taken together, these results do not support a role for IKK-mediated phosphorylation as means of regulating the activity of REL in vivo.;Results in this thesis also demonstrate that the histone acetyltransferases p300 and CREB-binding protein (CBP) can interact with the REL transactivation domain in vitro and in vivo, and can enhance REL's transactivation ability. p300 interacts with REL in both REL-transformed chicken spleen cells and in the diffuse large B-lymphoma cell line RC-K8, in which REL is constitutively active and required for proliferation. Furthermore, it is shown that only a C-terminally truncated form of p300 is expressed in RC-K8 cells. This is the first report of a p300 truncation in B-cell lymphoma. A model is presented for how mutations affect the REL/NF-kappaB pathway in the RC-K8 B-lymphoma cell line. Taken together, these results suggest a role for p300 in REL-mediated oncogenic activity.