Matthew Hayes

IDENTIFICATION OF DIS-3 (DISJUNCTION IN MEIOSIS) AS A NOVEL DIRECT TARGET OF ERK2. Matthew Hayes¹, Swathi Arur², Tim Schedl², Biology Department, Washington University, St. Louis, MO¹; Genetics Department, Washington University Medical School, St. Louis, MO.²

ERK2 (extra signal regulated kinase 2) is the terminal kinase in signaling cascades regulating proliferation, development and homeostasis in animals. Finding novel substrates of ERK is an active field of research because the inappropriate activation of ERK2 is known to contribute to tumorgenisis. In yeast, dis-3 was identified as a mitotic control protein. We identified dis-3 as a genetic downstream effector of ERK2 signaling involved in oocyte growth control in C. elegans germline development. Using genetics we inferred that dis-3 is negatively regulated by ERK2 to mediate oocyte growth.

To next assess whether DIS-3 is indeed a direct target of ERK2, the dis-3 cDNA was amplified from a region 1500bp to the C-terminal end of the gene and analyzed. This region contained a putative ERK2 phosphorylation site, at position 858 in the peptide, and an ERK2 docking domain. Two constructs, wildtype and S858A mutant, were generated using PCR and were cloned into the pTrcHis-1 vector and transformed into Escherichia coli. The goal of the experiment was to determine whether wildtype DIS-3 was indeed a substrate for phosphorylation by ERK2. In vitro the phospho-acceptor dead mutant will be used to test whether S858 is the phospho-acceptor used by active ERK2 enzyme for phosphorylation.

The E. coli containing the wildtype construct was grown and induced to produce DIS-3 protein. Purified protein was assayed as a substrate for active ERK2 enzyme in a kinase assay. The result of this assay confirmed that DIS-3 is a direct target of ERK2.

Once the mutant protein is purified a similar assay will be performed. This assay will test for loss of phosphorylation associated with the loss of this phospho-acceptor site and identify the exact phosphorylation residue used. Loss of phosphorylation will confirm that Serine 858 is the site of ERK2 phosphorylation on DIS-3.

In this study, we have identified DIS-3 as a direct target of ERK2, involved in oocyte growth and differentiation in C. elegans.