Regulation of myocyte enhancer factor 2: An investigation by mass spectrometry

Proteins are the primary machinery that determine cell function. Understanding proteins, and the networks that they function in, is the major challenge of the ‘proteomics’ revolution. Advances in this field will pioneer new approaches in medicine and further our knowledge of physiology.; Although all of the somatic cells in our body contain the same DNA sequence, we are made up of many different cells, all with very specific functions. Proteins, the way they interact, and how they are chemically modified, determine the immediate function of the cell and how it will respond to environmental cues. From the growth of a muscle cell to the development of neuronal cells, their responses are determined by what proteins are present and how they are modified.; The modern study of proteins benefits enormously from the use of mass spectrometry. Whether for protein identification, quantification, or discovering post-translational modifications, mass spectrometry has become the tool of choice for many researchers. Myocyte enhancer factor 2 (MEF2) is a highly regulated transcription factor that plays a diverse and critical role in gene expression of neurons, muscle, and immune cells (T cells), and thus presented an excellent opportunity to develop mass spectrometric based techniques. The pursuit of this aim involved the assembly of a mass spectrometer to identify our first unknown protein, and the development of new techniques for mass spectrometric based automated peptide sequencing. With high resolution mass spectrometric techniques in place we required biochemical tools for the study of mammalian transcription factors. Our development of tandem affinity purification (TAP) in mammalian cells gave us the necessary tools to combine mass spectrometry with the study of muscle development. The culmination of this work is represented by the application of these techniques to the analysis of the phosphorylation status of MEF2.; Tandem affinity purification was established in yeast by Seraphin's group as a method for purifying intact protein complexes. This peptide tag takes advantage of two separate non-denaturing purification steps (Protein A and calmodulin binding peptide). Contaminating proteins that can not be diluted away during the first step are removed during the second round of purification. Since the purifications utilise different methods of binding (Protein A with IgG vs. Ca++ dependent calmodulin binding) the contaminants from the first round of purification do not bind during the second round. We applied this technique to purify MEF2 from mammalian cells. By co-expressing TAP-MEF2A with MEF2C (a known interaction) we were able to co-purify both proteins, demonstrating the efficacy of this technique in mammalian cells.; The MEF2A purified from mammalian cells was found to contain post-translational modifications, unlike MEF2A expressed in bacterial cells. This included the identification of a novel phosphoacceptor site that regulates MEF2 stability, the identification of previously uncharacterised p38 MAPK phosphoacceptor sites, and the discovery of a protein kinase CK2 phosphoacceptor site that required p38 MAPK activity to be phosphorylated. By using tandem affinity purification combined with mass spectrometry we were able to document several novel aspects of MEF2 regulation. We used tandem affinity purification and mass spectrometry to characterise several novel aspects of MEF2 function. (Abstract shortened by UMI.)...