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REGULATION OF CDX2/3 TRANSCRIPTIONAL ACTIVITY BY P38 MAPK-DEPENDENT PHOSPHORYLATION
A Cartier, S Mongrain, N Rivard, C Asselin
Département d’anatomie et biologie cellulaire, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec
INTRODUCTION: Enterocyte differentiation is mediated in part by the transcription factor CDX2/3. We previously demonstrated that the p38 MAPKinase positively regulates CDX2/3. The aim of this study was to identify putative MAPK phosphorylation sites responsible for CDX2/3 transcriptional regulation.
METHODS: Site-directed mutagenesis of serines (S/P MAPK motif) into alanine or aspartic acid, and deletions of small N-terminal domains were performed. DNA-binding analysis of the various mutants was assessed by electrophoretic mobility shift and supershift assays. Interaction between the p38 MAPK and CDX2/3 mutants was determined by GST-CDX2/3 fusion protein pull-down assays. Phosphorylation by purified p38 and ERK1/2 MAPKs was evaluated by in vitro kinase assays of GST-fusion proteins. Transcriptional activity was determined by luciferase assays with the SI-luc and Lph-luc constructs, in HEK293 and human colon carcinoma Caco-2/15 cell lines. Expression of CDX2/3 targets was analyzed by RT-PCR and Northern blot of total RNAs isolated from IEC-6 intestinal epithelial cells stably expressing CDX2/3 mutants delivered by retroviral infection.
RESULTS: 1) CDX2/3 amino acids 61 to 76 were needed for p38 MAPK interaction. 2) A combination of S60A and S156A mutations dramatically decreased CDX2/3 phosphorylation. 3) CDX2/3 transcriptional activity was impaired by combinations of site-specific mutations in alanine or aspartic acid, in transient luciferase assays. 4) Site-specific mutants differentially impaired the expression of putative CDX2/3 targets, such as HNF4?, A2M, Myosin VIIb and Myosin XV.
CONCLUSION: MAPKs, including the p38 MAPK, regulate CDX2/3 transcriptional activity and function through multiple S/P motifs.
Supported by NSERC, CCFC and CIHR