Protein kinases play a key role in the regulation of metabolic activities in eukaryotic cells. A large number of protein kinases have been isolated and characterized, but we are still in the early stages of understanding the ramifications of phosphorylation on the target proteins. Better tools are needed to study protein kinases and the products of their action. We propose to design and manufacture a new generation of protein kinase substrates that will facilitate the analysis of these key enzymes. These new substrates will enable the simultaneous assay of multiple protein kinases and will help to characterize newly-discovered ones. The substrates consist of artificial proteins with a constant N-terminus tagged for ease of purification. A variable central region that is a polymer of a specific decapeptide and a specific C-terminal-portion that is an optimal substrate for a protein kinase. The only phosphorylatable site will be in this C-terminal region. By linking a specific C-terminal region to a specific length of the central region, many different kinase substrates can be generated that can all be displayed simultaneously by SDS-PAGE. These artificial proteins may also be valuable as markers in the low molecular weight range.
Will lead to improved substrates for the detection and characterization of protein kinases. As a byproduct, superior protein molecular weight markers may also emerge.
