271910 Engineered Monobody Inhibitors of Erk-2 Dependent Signaling

Wednesday, October 31, 2012: 10:00 AM
Westmoreland West (Westin )
Jasdeep Mann1, Anne-Fleur Stephan1, Jordan Wood2, Denise Ferkey2, E. Manolis Tzanakakis1 and Sheldon Park1, (1)Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, NY, (2)Biological Sciences, State University of New York at Buffalo, Buffalo, NY

The phosphorylation networks are central to intracellular signaling and orchestrate a broad range of cellular responses to environmental cues. The mitogen activated protein kinases (MAPK) form evolutionarily conserved pathways that are activated by stimuli at the cell surface and induce changes in gene expression. In human, there are at least four MAPK pathways, including the Erk1/2, p38, JNK, and Erk5 pathways, that mediate cellular responses to growth factors, inflammatory cytokines, and stress signals, and regulate embryogenesis, and cell fate determination. All MAPK pathways are organized in a three tier system, in which a MAPK is phosphorylated by a dual specificity MAPK kinase, or MEK, which is in turn phosphorylated by a MEK kinase (MEKK). Dysregulation of MAPK pathways is associated with various diseases, including cancer, diabetes, neurological and inflammatory diseases. Therefore, engineered MAPK inhibitors would be useful tools for studying the molecular basis of signaling in model organisms and for developing therapeutics against overactive signaling networks. In the current study, we describe the engineering of monobody inhibitors that selectively block the interaction between Erk-2 and its in vivo substrates, activators, and negative regulators. MAPKs use the docking of a D-motif peptide found on their interactors and conserved “CD” docking domain to confer specificity of interaction and increase the efficiency of signaling. Since the docking interaction is essential for the function of Erk-2, the designed monobody should efficiently reduce the efficiency of Erk-2 activation and its catalytic efficiency. We screened a combinatorial fibronectin type III (FN3) monobody library expressed on the yeast surface using magnetic and fluorescence activated cell sorting. To isolate the monobodies that specifically target the CD domain, affinity maturation was alternated with a negative sort using mutant Erk-2 containing mutations in the docking site. The clones identified from the sort bind Erk-2 at the docking site with low nM affinity and inhibit its phosphorylation by MEK2 as well as phosphorylation of the transcription factor Elk1. Some of the clones exhibit high target specificity and can discriminate against p38 and JNK with > 100 fold selectivity. The inhibitors strongly inhibit the Erk-2 pathway in transfected HEK293 cells and affect the subcellular location of Erk-2 by disrupting its interaction with upstream kinases. Finally, we show that the monobodies can modulate the physiological processes in yeast and worms by inhibiting the activity of Erk-2 orthologs in each organism, demonstrating that the engineered inhibitors are useful in a variety of cellular context.

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