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Marina Jeyasingham
Research Assistant Professor |
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Protein phosphorylation is arguably the most important mechanism for the regulation of a number of metabolic processes. This covalent modification is catalyzed by a large family of enzymes known collectively as protein kinases, which invariably use ATP to phosphorylate protein substrates. Nevertheless, the higher energy of hydrolysis of phosphoenolpyruvate (PEP), a ubiquitous glycolytic intermediate, gives this compound a greater potential than ATP to serve as a phosphoryl group donor for protein phosphorylation; and although this potential has been realized to some extent in prokaryotes, the presence of an analogous system in eukaryotes has not been demonstrated. Results from our laboratory have shown that a PEP-requiring protein kinase activity that is independent of ATP is present in several tissues, including skeletal muscle, heart, liver and brain. Further studies of this activity have identified a number of proteins that are apparently directly phosphorylated by PEP. The aims of our ongoing research are to identify other protein substrates in these tissues and to isolate and characterize their associated PEP-dependent protein kinase(s). Our ultimate goal is to clarify the role of these enzymes in each tissue, thus delineating a potentially unique signaling cascade linking glycolysis (and the energy status of the cell) with other metabolic pathways.
To accomplish these objectives a variety of approaches have been applied, including traditional biochemical methodologies such as radiometric and spectrophotometric assays, protein purification using a battery of column chromatography supports and general techniques of protein chemistry. Moreover, the recent rapid developments in proteomics have increased the arsenal of tools available to us. Two-dimensional polyacrylamide gel electrophoresis combined with mass spectrometry, peptide mass fingerprinting and database mining have provided fast, sensitive and routine strategies for the identification of these protein substrates.
