| Adequate nutrition is of vital importance for all living individuals. It is even more important for infants, as they need nutrition for growth. Damage of the intestinal absorptive surface, resulting in inadequate absorption of nutrients, is very prevalent in children (various congenital and neonatal disorders, infectious enteropathies, celiac disease, inflammatory bowel disease and chemo- or radiation therapy). It can cause serious complications. Knowledge of the molecular regulation of intestinal cell renewal and differentiation can provide new strategies for treating or preventing a variety of diseases that affect the gastrointestinal surface. Our research focuses on Cdx2. Cdx2 is an intestine-specific transcription factor. A growing number of studies show that Cdx2 is important in a broad range of functions, ranging from early differentiation and growth to maintenance of the intestinal epithelial lining. Cdx2 function is established after post-translational modification. Protein phosphorylation is the post-translational modification of choice for situations where rapid change of transcriptional activity is required. We recently found that the functional domain (activation domain) of Cdx2 at position serine 60 is a target for mitogen-activated protein kinase (MAPK) phosphorylation. Phosphorylated Cdx2 was primarily found in proliferating enterocytes, while non-phosphorylated Cdx2 was mainly found in the differentiated enterocytes. Interestingly, phosphorylation of serine 60 reduced Cdx2 activity profoundly. The present project aims to test why the phosphorylation status of serine 60 in the Cdx2 activation domain functions as an important physiological switch for Cdx2 activity and thus for intestinal gene expression, modulating both the growth and the differentiation of enterocytes. Cdx2 function specified by its phosphorylation status will be addressed in this project. Serine 60 phosphorylation will be modified in cellular models using specific stimuli and inhibitors of the respective phosphorylation pathway. Confirmation of the effect of these modifications will be tested using constitutive "phosphorylated" or "non-phosphorylated" Cdx2 mutants in intestinal cell lines. To delineate the mechanism by which the status of serine 60-phosphorylation modifies Cdx2 function, the transactivation complex will be analyzed by identifying the partners in the transcriptional complex using affinity purification of Cdx2 from cells that either do or do not phosphorylate the serine 60 position. The in vivo effect of constitutive "phosphorylated" or "non-phosphorylated" Cdx2 mutants will then be tested in mice in whom the serine 60 is substituted by these respective mutants. |
