Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins

An emerging theme in transforming growth factor-beta (TGF-beta) signalling is the association of the Smad proteins with diverse groups of transcriptional regulatory proteins. Several Smad cofactors have been identified to date but the diversity of TGF-beta effects on gene transcription suggests that interactions with other co-regulators must occur. In these studies we addressed the possible interaction of Smad proteins with the myocyte enhancer-binding factor 2 (MEF2) transcriptional regulators. Our studies indicate that Smad2 and 4 (Smad2/4) complexes cooperate with MEF2 regulatory proteins in a GAL4-based one-hybrid reporter gene assay. We have also observed in vivo interactions between Smad2 and MEF2A using co-immunoprecipitation assays. This interaction is confirmed by glutathione S-transferase pull-down analysis. Immunofluorescence studies in C2C12 myotubes show that Smad2 and MEF2A co-localise in the nucleus of multinuclear myotubes during differentiation. Interestingly, phosphoacceptor site mutations of MEF2 that render it unresponsive to p38 MAP kinase signalling abrogate the cooperativity with the Smads suggesting that p38 MAP Kinase-catalysed phosphorylation of MEF2 is a prerequisite for the Smad-MEF2 interaction. Thus, the association between Smad2 and MEF2A may subserve a physical link between TGF-P signalling and a diverse array of genes controlled by the MEF2 cis element.