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 transcriptio nal regulatory proteins. Several Smad cofactors have been identified to dat e but the diversity of TGF-beta effects on gene transcription suggests that interactions with other co-regulators must occur. In these studies we addr essed the possible interaction of Smad proteins with the myocyte enhancer-b inding factor 2 (MEF2) transcriptional regulators. Our studies indicate tha t Smad2 and 4 (Smad2/4) complexes cooperate with MEF2 regulatory proteins i n a GAL4-based one-hybrid reporter gene assay. We have also observed in viv o interactions between Smad2 and MEF2A using co-immunoprecipitation assays. This interaction is confirmed by glutathione S-transferase pull-down analy sis. 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 unre sponsive to p38 MAP kinase signalling abrogate the cooperativity with the S mads suggesting that p38 MAP Kinase-catalysed phosphorylation of MEF2 is a prerequisite for the Smad-MEF2 interaction. Thus, the association between S mad2 and MEF2A may subserve a physical link between TGF-P signalling and a diverse array of genes controlled by the MEF2 cis element.