Structural studies of novel phosphorylation sites in Erk MAP kinases

ERK2, a prominent member of the mitogen activated protein kinase (MAPK) family, is a key protein of mammalian cells, activated in response to a variety of extracellular signals such as growth factors, and other stresses. Like all the MAPK`s, ERK2 is activated via dual phosphorylation of a Thr-Xaa-Tyr motif, by the upstream activator MAPK kinase (MEK/MAPKK). After activation ERK phosphorylates a diverse panel of substrates in both the cytoplasm and the nucleus. Thus, regulating many cellular processes including: proliferation, differentiation, development and more. Enhanced activation of ERK2 has been found in numerous cancers, inflammatory and neurodegenerative diseases. ERK2`s apparent role in malignant diseases further emphasizes the need for a better understanding of its activation pathway and activation mechanism in particular. It was shown that the R65S mutation exhibit significant intrinsic activity. MS analysis of the R65S mutants identified a new phosphorylation site Thr188 that was also involved in cardiac hypertrophy. Mutants of Thr188 to either Ala/ Glu/Asp abolished the activity of ERK2. In our study we have solved the three-dimensional structures of T188Ala/Glu/Asp+R65S to high resolution to obtain better understanding of the structural basis of phosphorylation of Thr188. All of the structures maintained the overall MAPK topology but showed different conformation in the L16 region, a unique extension in the MAPK family. Further analysis for the L16 conformational change and crystallization of the mono-phosphorylated T188 ERK2 are in progress.