DNA Polymerase Epsilon Subunit B is a novel conserved substrate of active ERK2

Amanda Hay and Tim Schedl

Department of Genetics, Washington University School of Medicine

ERK MAP kinase is the terminal kinase of the Receptor Tyrosine Kinase signaling cascade, and thus controls many aspects of development in C. elegans and humans.  ERK MAP kinase executes normal development by phosphorylation of target protein products. A key question of our lab is: What are the target proteins being phosphorylated by active ERK to execute normal germline development in C. elegans? My mentor, Swathi Arur, has identified C. elegans DNA polymerase epsilon subunit B as a genetic interactor of MPK-1 ERK2. It is involved in the regulation of normal oocyte growth. The primary focus of my summer research was to assay whether this gene, F08B4.5, is an in vitro substrate of active ERK2.

To do this, I amplified F08B4.5 from C. elegans cDNA and inserted it into a bacterial expression vector. The clone with the correct insert was checked for mutations by sequencing. I incubated this bacterial colony and induced production of recombinant proteins, which were then affinity purified by NI-NTA resin.  The purified protein was eluted and tested in an in vitro kinase assay using activated murine active ERK2.  The purified recombinant F08B4.5 was found to be phosphorylated by active ERK2 in vitro. Thus, we found that F08B4.5 is a novel target of ERK2. Given the conserved nature of the ERK signaling pathway, and of F08B4.5 in humans, it is very likely that DNA polymerase epsilon subunit B may be regulated by ERK2 in higher organisms in much the same manner as it is in C. elegans.