The mitogen-activated protein kinase pathway regulates the subcellular localization and function of FOXM1
"The Mitogen-Activated Protein Kinase Pathway Regulates the Subcellular Localization and Function of FOXMl"
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 MI (FOXMI) is ubiquitously expressed in proliferating cells and is believed to play a role in the regulation of cell cycle progression. FOXMI knockout mice die at perinatal period with heart and liver cells severely polyploid. However, transgenic mice overexpressing FOXMI do not have apparent phenotype but liver and lung regenerations are accelerated following damages to these organs. Tissue sections of these FOXMI-overexpressing mice indicate that FOXMI is retained in cytoplasm in quiescent cells and upon stimulation by mitogenic signals FOXMI becomes nuclear localized. Nuclear localization of FOXMI is essential for its growth-promoting effect.
In this study, I attempted first to investigate the cell cycle phase during which FOXMI enters nucleus. Antibody staining in synchronized human foreskin fibroblast hTERT-BJI cells and mouse embryonic fibroblast NIH 3T3 cells indicates that
FOXMl translocates from cytoplasm into nucleus during late S phase of the cell cycle. Reverse transcription-polymerase chain reactions suggest that FOXMlC 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 NllI 3T3 cells reveals that FOXMl C contains two putative nuclear localization signals essential for its nuclear import. FOXMlC is not subjected to Crml-dependent nuclear export because it is refractory to Leptomycin B treatment. Eps8, an actinassociating protein, interacts strongly with FOXMl C in coimmunoprecipitation assay. The predominant cytoplasmic localization of Eps8 suggests that FOXMl may be retained cytoplasmically via interaction with Eps8.
The second goal of this study was to understand the mitogenic dependency of FOXMl function by investigating whether nuclear translocation of FOXMl requires active Mitogen-Activated Protein Kinase (MAPK) signaling. Treatment of BJ1 cells with MAPK signaling activator aurintricarboxylic acid (ATA) triggers nuclear translocation of FOXMl and this effect is abolished by pre-treatment of specific MAPK kinase inhibitor U0126. U0126 treatment also blocks nuclear translocation of FOXMl 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 FOXMl C strongly trans activates the FOXMl target Cyclin B 1. When dominant negative MAPK kinase or dominant negative FOXMlC is used for coexpression, the activating effect is diminished.
Consistent with regulation of FOXMl function by the MAPK signaling pathway, sequence analysis identifies two putative ERK phosphorylation sites (serine331 and serine-704) and immunoprecipitated FOXMl could react with an anti-
phospho serine 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 FOXMIC as suggested by luciferase assays. Interestingly, study of two other FOXMI isoforms, FOXMIA and FOXMIB, reveals differences in their response to MAPK signaling and subcellular localization.
In summary, I have provided evidence to support the regulated nuclear translocation of FOXMI and its regulation by the MAPK pathway. This provides important insight into the regulation ofFOXMI function and its role in the control of cell cycle progression.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:protein kinases transcription factors cellular signal transduction genetic regulation
Date of Publication:01/01/2004