When molecular signals governing cellular proliferation in cells go awry, the progression of cancer occurs within once healthy cells. The MAPK pathway is one of the most common cell proliferation signaling cascade and is comprised of protein kinases Ras-Raf-MEK-ERK. These proteins sustain cellular communication through a cascade of phosphorylation events, which modulate the activity of each protein in the pathway. We are particularly interested in MEK1 for its key role in the MAPK pathway. Our understanding of the structure-function relationships resulting from the site-specific protein phosphorylation of MEK1 has been limited by the lack of technology to produce a homogeneous population of phospho-MEK1 at high yields. Since production of the phosphoprotein has not been robust, the crystal structure for phospho-MEK1 has not been solved. The past decade has brought substantial advancements in the biotechnology field, enabling us to produce phosphoproteins at high yields through cell-free protein synthesis (CFPS). CFPS enables a technology platform that utilizes genetically engineered protein synthesis machinery by exploiting the expanded genetic code to site-specifically phosphorylate MEK. In parallel, we have developed a computation model of phospho-MEK1 that simulates the phosphorylation of two key serine residues (SER-218 and SER-222) in the wildtype MEK crystal structure. This phosphorylated model is then be used in a molecular dynamics simulation to refold the structure into a model of a reasonable phospho-MEK1 conformation. When paired with a computational structural model for phospho-MEK, the ability to manufacture phosphorylated active MEK1 enables us to ask previously intractable questions about MEK1’s structure and function. Here we report our efforts to develop a biotechnology platform that integrates the production of phospho-MEK1, with a computationally-guided high-throughput activity assay in a one-pot system. We accomplish this through the optimization of existing immunoassays for MEK1 activity that examine the activation of the substrate protein, ERK for integration with CFPS. These efforts will lead to an integrated technology in which computationally-guided MEK1 and ERK mutant libraries can be used to elucidate structure-function relationships with rapid design-build-test cycles.

Elucidating structure-function relationships of the phosphorylated human protein MEK1 through the integration of biotechnologies

253rd National Meeting (2017)

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