Intracellular signaling networks are primarily controlled by a complex array of covalent protein modifications that either reversibly or irreversibly alter protein function, multimerization, location, or all three. The most well studied protein modification is phosphorylation, yet our understanding of how specificity is maintained in protein kinase phosphorylation cascades is poorly understood. The hypothesis guiding this study is that domains on signaling proteins mediate assembly into specific complexes via interaction with relatively short sequence motifs on target proteins. Additional fidelity is derived by the specificity of protein kinase catalytic domains for unique substrate motifs. Thus, subtle differences between the motifs recognized by the catalytic pocket of protein kinases and/or differences between the motifs recognized by protein interaction domains outside the catalytic pocket can ultimately explain how individual protein kinases phosphorylate unique sites in vivo. Peptide library screening techniques developed in this laboratory over the past 12 years make it feasible to rapidly deduce the motifs recognized by individual protein interaction domains and motifs phosphorylated by protein kinases.
The aims of this grant are: 1) to use a novel peptide library approach to define optimal substrates for protein kinases implicated in cell growth pathways; 2) to use phosphopeptide motif mixtures to identify new phosphoprotein binding domains; 3) to use a similar approach to identify protein domains that bind to other types of modified amino acids; and 4) to further develop our Internet-based Scansite program for predicting sites of protein modification and protein interactions based on sequences in protein databases.
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