MAPK (mitogen-activated protein kinase) cascades are involved in a variety of cellular functions including proliferation, differentiation, and apoptosis. Upon activation by any of a number of mechanisms, Mekk (a MAPK kinase kinase) activates Mkk (a MAPKK or Sek), which in turn activates SAPK (a MAPK). The MAPK is then translocated from the cytoplasm to the nucleus, where it directly regulates the activity of transcription factors controlling the expression of a number of genes. Recently, we observed Mekk1 and its constitutively active fragment CA-Mekk1 (1174-1493 aa) were translocated to the nucleus when overexpressed in HEK293T, COS, and HCT cells and affected transcription of the Pkd1 (Polycystic kidney disease) gene and possibly other genes via the anti-tumor protein, p53. As such, we have begun to unravel a novel mechanism of transcriptional regulation. Thus, with a broad goal to understand the novel mechanism of Mekk1-p53 mediated transcriptional regulation of the Pkd1 and other genes, we propose to accomplish the following four Specific Aims:
Aim 1 : Identify fragment(s) of Mekk1 translocating to the nucleus and determine their mechanism of nuclear translocation.
Aim 2 : Characterize the p53-Mekk1 binding mechanism by: (a) identifying the binding site if it is direct, or (b) identifying the intermediate partners, if it is indirect.
Aim 3 : Investigate the mechanism of p53 elevation.
Aim 4 : Perform ChIP-Seq analysis to identify genome-wide binding sites for Mekk1, p53 and both. We will use a variety of biochemical and cellular/molecular biological approaches as well as the latest cutting edge technique, ChIP-Seq. These studies will not only improve our understanding of transcriptional regulation by both p53 and Mekk1, but may also uncover the mechanism of the Mekk1-p53 pathway. Thus, the answers sought should have both fundamental significances and should provide information leading to future therapeutic approaches. The research will engage undergraduates at Northwest Missouri State University (NWMSU), as well as talented high school students in the Missouri Academy of Science, Math, and Computing (MASMC) housed on the Northwest campus. Training received in this project will prepare students for entry into science programs and/or scientific professional careers. Thus, the project will (i) provide undergraduate and talented high school students benefits of exposure to and participation in biomedical research, (ii) strengthen the research environment at NWMSU, and (iii) give the PI an opportunity to continue meritorious research. Furthermore, collaborations with investigators at a research-intensive institution (University of Kansas Medical Center) will ensure the infusion of the latest methodology and information into the biochemistry/cell biology curricula. Thus, the project will advance discovery and understanding while also promoting research, training and learning.
Recently, we unraveled a novel mechanism in which nuclear translocation of Mekk1 and its interaction with p53 leads to transcriptional repression of PKD1, independent of the MAPK phosphorylation cascade. The mechanism was previously unknown. This proposal will study the mechanism in detail and will seek to determine how Mekk1, a membrane-associated protein, translocates to the nucleus and interacts with p53;and will utilize cutting-edge genome-wide approaches to identify other genes that might be under regulation of this Mekk1-p53 pathway.
