Regulation of ERK3/4 Function via Specific Protein-Protein Interactions

Abstract

The mitogen-activated protein kinase 5 (MK5) binds to extracellular signal-regulated kinase 3 and 4(ERK3 and ERK4) through a unique FRIEDE motif in ERK3/4. 100 amino acids C-terminal in MK5 is sufficient for this binding. MK5s subcellular localization and activity is regulated by the atypical MAP kinase ERK3 and ERK4. In this project we wanted to express and purify full-length and different domains of MK5 and use it to gain structural information of the kinase to further reveal how it binds to ERK3 and ERK4 at the atomic level. Recombinant technics for generation of these proteins together with functional assays to assess the activity of MK5 have been used. Optimal protocols for expression and purification of the kinase was developed. Finally we used a GST pulldown assay to show that the amino acid domain amino acid domain 372-473 of MK5 are able to bind to ERK4 in extracts from mammalian cells.