| The transforming growth factor-beta (TGFbeta) signaling pathway affects a diverse range of biological functions including differentiation, cell growth arrest, apoptosis, and epithelial-to-mesenchymal transition (EMT). Stimulation of cells with TGFbeta enables the activated TGFbeta serine/threonine kinase receptor complex to phosphorylate Smad2 and Smad3, allowing Smad2/3 to oligomerize with Smad4 and translocate into the nucleus. Once in the nucleus, the Smad complex binds specific promoters and recruits coactivators such as p300/CBP to initiate transcription of TGFbeta target genes.p300/CBP directly interacts with the c-termini of Smad2 and Smad3 in a ligand dependent manner to increase the transcriptional activity of the Smad complex. This interaction was originally thought to function to modify chromatin into its acetylated "open" conformation. However the discovery that p300/CBP can also act to acetylate non-histone proteins suggests an alternate role. Acetylation of non-histone proteins can affect everything from protein stability, binding ability to DNA and other proteins, and protein localization. This dissertation describes the findings that Smad2 can be acetylated in a p300 dependent manner while Smad3, a protein with 93% sequence identity to Smad2, cannot. This difference is due to the presence of a 10 amino acid insert termed the GAG region found only in Smad2. A combination of mutational analysis and mass spectrometry identified lysines 19, 20, and 39 as acetylation sites. Lysine to arginine mutations were used to determine the biological consequence of Smad2 acetylation. The Smad2 lysine to arginine mutants demonstrated decreased transcriptional activity in Smad2-deficient mouse embryonic fibroblasts (MEF) that translated into a loss of TGFbeta stimulated cell growth arrest. The Smad2 mutants demonstrated a loss in nuclear retention in cells treated with TGFbeta. Treatment with the TGFbeta receptor inhibitor SB-431542 suggested acetylation of Smad2 contributes to its nuclear localization by decreasing the rate of export of the protein upon stimulation with TGFbeta.These studies detailed in this thesis reveal a novel post-translational modification of Smad2 that contributes to its transcriptional activity by altering the protein's subcellular localization, retaining the protein in the nucleus upon TGFbeta stimulation through a decrease in nuclear export. |
