This dissertation, "The Mitogen-activated Protein Kinase Pathway Regulates the Subcellular Localization and Function of FOXM1" by Yam-man, Richard, Ma, 馬蔭民, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled "The Mitogen-Activated Protein Kinase Pathway Regulates the Subcellular Localization and Function of FOXM1" Submitted by Ma Yam Man, Richard for the degree of Master of Philosophy at The University of Hong Kong in August 2003 The forkhead box transcription factor M1 (FOXM1) is ubiquitously expressed in proliferating cells and is believed to play a role in the regulation of cell cycle progression. FOXM1 knockout mice die at perinatal period with heart and liver cells severely polyploid. However, transgenic mice overexpressing FOXM1 do not have apparent phenotype but liver and lung regenerations are accelerated following damages to these organs. Tissue sections of these FOXM1-overexpressing mice indicate that FOXM1 is retained in cytoplasm in quiescent cells and upon stimulation by mitogenic signals FOXM1 becomes nuclear localized. Nuclear localization of FOXM1 is essential for its growth-promoting effect. In this study, I attempted first to investigate the cell cycle phase during which FOXM1 enters nucleus. Antibody staining in synchronized human foreskin fibroblast hTERT-BJ1 cells and mouse embryonic fibroblast NIH 3T3 cells indicates that FOXM1 translocates from cytoplasm into nucleus during late S phase of the cell cycle. Reverse transcription-polymerase chain reactions suggest that FOXM1C is the unique isoform expressed in NIH 3T3 cells and most mouse tissues, and it is probably subjected to the above regulation. Expression of HA-tagged and GFP-tagged mutants in NIH 3T3 cells reveals that FOXM1C contains two putative nuclear localization signals essential for its nuclear import. FOXM1C is not subjected to Crm1-dependent nuclear export because it is refractory to Leptomycin B treatment. Eps8, an actin- associating protein, interacts strongly with FOXM1C in coimmunoprecipitation assay. The predominant cytoplasmic localization of Eps8 suggests that FOXM1 may be retained cytoplasmically via interaction with Eps8. The second goal of this study was to understand the mitogenic dependency of FOXM1 function by investigating whether nuclear translocation of FOXM1 requires active Mitogen-Activated Protein Kinase (MAPK) signaling. Treatment of BJ1 cells with MAPK signaling activator aurintricarboxylic acid (ATA) triggers nuclear translocation of FOXM1 and this effect is abolished by pre-treatment of specific MAPK kinase inhibitor U0126. U0126 treatment also blocks nuclear translocation of FOXM1 in synchronized BJ1 cells during late S phase of the cell cycle, suggesting that MAPK signaling is required for such regulation. Using luciferase assays, this study further shows that coexpression of constitutively active MAPK kinase and FOXM1C strongly transactivates the FOXM1 target Cyclin B1. When dominant negative MAPK kinase or dominant negative FOXM1C is used for coexpression, the activating effect is diminished. Consistent with regulation of FOXM1 function by the MAPK signaling pathway, sequence analysis identifies two putative ERK phosphorylation sites (serine- 331 and serine-704) and immunoprecipitated FOXM1 could react with an anti- phosphoserine antibody. The predicted phosphorylation sites are important for MAPK regulation as substitution of serine-331 and serine-704 with alanines completely abrogates the MAPK-induced activation of FOXM1C as suggested by luciferase assays. Interestingly, study