MEK1 is an ideal drug target for the therapeutic treatment of cancerous mutations that occur in the MAPK phosphorylation cascade, one of the most common cell proliferation signaling pathways. The study of the specific phosphorylation event between MEK1 and its only known substrate ERK2 has previously been limited due to the difficulty in obtaining the active phosphorylated form phospho-MEK1. Our study aims to overcome this barrier by utilizing the novel technology of the expanded genetic code to produce site-specifically phosphorylated proteins. In order to elucidate the structure-function relationships of MEK1, we are pursuing a hypothesisdriven, structure-guided approach based on conserved kinase motifs for the creation of variants using site-specific mutagenesis. We will optimize immunoassays in order to quantify the enzymatic activity of both the wild-type and mutant forms of MEK1. We will subsequently characterize the enzyme kinetics of MEK1 mutants to deduce the significance of specific residue interactions in the phosphorylation event. Our findings will be the first to establish kinetic parameters for the wild-type phospho- MEK1, further the understanding of MEK1’s structure-function relationships in the cell signaling event, and enable the development of more effective MEK1 inhibitors for the treatment of cancerous mutations.
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